Diphenylmethyl compounds useful as muscarinic receptor antagonists

ABSTRACT

This invention provides compounds of formula I:  
                 
 
wherein a, b, c, e, m, R 1 , R 2 , R 3 , R 4a , R 4b , R 5 , R 6 , R 7  and R 8 are as defined in the specification. The compounds of formula I are muscarinic receptor antagonists. The invention also provides pharmaceutical compositions containing such compounds, processes and intermediates for preparing such compounds and methods of using such compounds to treat pulmonary disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/552,368, filed on Mar. 11, 2004; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel diphenylmethyl compounds havingmuscarinic receptor antagonist or anticholinergic activity. Thisinvention also relates to pharmaceutical compositions comprising suchdiphenylmethyl compounds, processes and intermediates for preparing suchdiphenylmethyl compounds and methods of using such diphenylmethylcompounds to treat pulmonary disorders.

2. State of the Art

Pulmonary or respiratory disorders, such as chronic obstructivepulmonary disease (COPD) and asthma, afflict many millions of peopleworldwide and such disorders are a leading cause of morbidity andmortality.

Muscarinic receptor antagonists are known to provide bronchoprotectiveeffects and therefore, such compounds are useful for treatingrespiratory disorders, such as COPD and asthma. When used to treat suchdisorders, muscarinic receptor antagonists are typically administered byinhalation. However, even when administered by inhalation, a significantamount of the muscarinic receptor antagonist is often absorbed into thesystemic circulation resulting in systemic side effects, such as drymouth, mydriasis and cardiovascular side effects.

Additionally, many inhaled muscarinic receptor antagonists have arelatively short duration of action requiring that they be administeredseveral times per day. Such a multiple-daily dosing regime is not onlyinconvenient but also creates a significant risk of inadequate treatmentdue to patient non-compliance with the required frequent dosingschedule.

Accordingly, a need exists for new muscarinic receptor antagonists. Inparticular, a need exists for new muscarinic receptor antagonists thathaving high potency and reduced systemic side effects when administeredby inhalation. Additionally, a need exists for inhaled muscarinicreceptor antagonists having a long duration of action thereby allowingfor once-daily or even once-weekly dosing. Such compounds are expectedto be particularly effective for treating pulmonary disorders, such asCOPD and asthma, while reducing or eliminating side effects, such asdry-mouth and constipation.

SUMMARY OF THE INVENTION

The present invention provides novel diphenylmethyl derivatives havingmuscarinic receptor antagonist or anticholinergic activity. Among otherproperties, compounds of the invention are expected to possess highpotency and reduced systemic side effects when administered byinhalation and to have a long duration of action.

Accordingly, in one of its composition aspects, the present inventionprovides a compound of formula I:

wherein:

-   -   each R¹ and R² are independently selected from (1-4C)alkyl,        (2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo,        —OR^(a), —SR^(a), —NR^(a)R^(b), —S(O)R^(c) and —S(O)₂R^(c);        where each R^(a) and R^(b) independently represents hydrogen,        (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl or (3-6C)cycloalkyl;        each R^(c) independently represents (1-4C)alkyl, (2-4C)alkenyl,        (2-4C)alkynyl or (3-6C)cycloalkyl; or two adjacent R¹ groups or        two adjacent R² groups are joined together to form        (3-6C)alkylene, (2-4C)alkylene-O— or —O— (2-4C)alkylene-O—;    -   a and b each independently are 0 or an integer of from 1 to 5;    -   each R³ independently is fluoro or (1-4C)alkyl;    -   c is 0 or an integer of from 1 to 3;    -   R^(4a) and R^(4b) are independently selected from hydrogen,        (1-4C)alkyl, and phenyl-(1-4C)alkyl; or R^(4a) and R^(4b)        together with the carbon atom to which they are attached form a        (3-6C)heterocyclic ring optionally containing one additional        heteroatom selected from nitrogen, oxygen or sulfur and wherein        the heterocyclic ring is unsubstituted or substituted with 1 or        2 substituents selected independently from (1-4C)alkyl and        fluoro;    -   e is 1 or 2;    -   m is 4, 5 or 6;    -   R⁵ is selected from hydrogen, (1-4C)alkyl, and (3-4C)cycloalkyl;    -   R⁶ is hydrogen or an unbranched (1-4C)alkyl; or R⁶ and R⁸ are        joined, together with the atoms to which they are attached, to        form a pyrrolidin-2-yl group;    -   R⁷ is selected from hydrogen, (1-6C)alkyl, (3-6C)cycloalkyl,        —CH₂Ar¹, —CH₂CH₂—OH and —CH₂CH₂—O-(1-4C)alkyl; wherein Ar¹        represents phenyl or (3-5C)heteroaryl, wherein the phenyl or        heteroaryl group is unsubstituted or substituted with from 1 to        3 substituents selected independently from halo, (1-4C)alkyl and        (1-4C)alkoxy; wherein each alkyl and alkoxy is optionally        substituted with from 1 to 3 fluoro substituents; and    -   R⁸ is hydrogen or (1-6C)alkyl; or R⁷ and R⁸ together with the        nitrogen atom to which they are attached form a pyrrolidin-1-yl,        piperidin-1-yl, morpholin-1-yl or thiomorpholin-1-yl group;    -   wherein each alkyl group in R¹, R², R³, R^(4a), R^(4b), R⁵, R⁶,        R⁷, R⁸ and R^(a-c) is optionally substituted with from 1 to 5        fluoro substituents;    -   or a pharmaceutically acceptable salt or solvate or stereoisomer        thereof.

In another of its composition aspects, this invention is directed to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of formulaI or a pharmaceutically acceptable salt or solvate or stereoisomerthereof. Such pharmaceutical compositions may optionally contain othertherapeutic agents. Accordingly, in one embodiment, this invention isdirected to such a pharmaceutical composition wherein the compositionfurther comprises a therapeutically effective amount of a steroidalanti-inflammatory agent, such as a corticosteroid; a β₂ adrenergicreceptor agonist; a phosphodiesterase-4 inhibitor; or a combinationthereof.

Compounds of this invention possess muscarinic receptor antagonistactivity. Accordingly, compounds of formula I are expected to be usefulfor treating pulmonary disorders, such as chronic obstructive pulmonarydisease and asthma.

Accordingly, in one of its method aspects, this invention is directed toa method for treating a pulmonary disorder, the method comprisingadministering to a patient a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or solvateor stereoisomer thereof.

Additionally, in another of its method aspects, this invention isdirected to a method of producing bronchodilation in a patient, themethod comprising administering to a patient a bronchodilation-producingamount of a compound of formula I or a pharmaceutically acceptable saltor solvate or stereoisomer thereof.

This invention is also directed to a method of treating chronicobstructive pulmonary disease or asthma, the method comprisingadministering to a patient a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt or solvateor stereoisomer thereof.

In another one of its method aspects, this invention is directed to amethod for antagonizing a muscarinic receptor in a mammal comprisingadministering to the mammal, a therapeutically effective amount of thecompound of formula I.

Since compounds of this invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools. Accordingly,in yet another of its method aspects, this invention is directed to amethod for using a compound of formula I or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof as a research toolfor studying a biological system or sample, or for discovering newchemical compounds having muscarinic receptor antagonist activity.

This invention is also directed to processes and novel intermediatesuseful for preparing compounds of formula I or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof. Accordingly, inanother of its method aspects, this invention is directed to a processof preparing a compound of formula I, the process comprising:

-   -   (a) reacting a compound of formula II with a compound of formula        III;    -   (b) reacting a compound of formula IV with a compound of formula        V;    -   (c) reacting a compound of formula II with a compound of formula        VI; or    -   (d) for a compound of formula I in which R⁶ represents hydrogen        or an unbranched (1-4C)alkyl group, reacting a compound of        formula VII with a compound of formula VIII;        to provide a compound of formula I; wherein compounds of formula        II-VIII are as defined herein.

In one embodiment, the above process further comprises the step offorming a pharmaceutically acceptable salt of a compound of formula I.In other embodiments, this invention is directed to the other processesdescribed herein; and to the product prepared by any of the processesdescribed herein.

This invention is also directed to a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof, foruse in therapy or as a medicament.

Additionally, this invention is directed to the use of a compound offormula I or a pharmaceutically acceptable salt or solvate orstereoisomer thereof, for the manufacture of a medicament; especiallyfor the manufacture of a medicament for the treatment of a pulmonarydisorder or for antagonizing a muscarinic receptor in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

In one of its composition aspects, this invention is directed to noveldiphenylmethyl compounds of formula I or pharmaceutically acceptablesalts or solvates or stereoisomers thereof. These compounds may containone or more chiral centers and therefore, this invention is directed toracemic mixtures; pure stereoisomers (i.e., enantiomers ordiastereomers); stereoisomer-enriched mixtures and the like unlessotherwise indicated. When a particular stereoisomer is shown or namedherein, it will be understood by those skilled in the art that minoramounts of other stereoisomers may be present in the compositions ofthis invention unless otherwise indicated, provided that the desiredutility of the composition as a whole is not eliminated by the presenceof such other isomers.

In particular, when e is 1, the compounds of formula I contain a chiralcenter at the carbon atom indicated by the symbol * in the followingpartial formula (shown without optional substituents for clarity):

In one embodiment of this invention, the carbon atom identified by thesymbol * has the (R) configuration. In this embodiment, it is preferredfor compounds of formula I to have the (R) configuration at the carbonatom identified by the symbol * or to be enriched in a stereoisomericform having the (R) configuration at this carbon atom. In anotherembodiment of this invention, the carbon atom identified by the symbol *has the (S) configuration. In this embodiment, it is preferred forcompounds of formula I to have the (S) configuration at the carbon atomidentified by the symbol * or to be enriched in a stereoisomeric formhaving the (S) configuration at this carbon atom.

The compounds of formula I also contain several basic groups (e.g.,amino groups) and therefore, the compounds of formula I can exist as thefree base or in various salt forms. All such salt forms are includedwithin the scope of this invention. Furthermore, solvates of compoundsof formula I or salts thereof are included within the scope of thisinvention.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds offormula I are included within the scope of this invention unlessotherwise specified.

The compounds of formula I, as well as those compounds used in itssynthesis, may also include isotopically-labeled compounds, i.e., whereone or more atoms have been enriched with atoms having an atomic massdifferent from the atomic mass predominately found in nature. Examplesof isotopes that may be incorporated into the compounds of Formula (I)include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O and ¹⁷O.

The nomenclature used herein to name the compounds of this invention isillustrated in the Examples herein. This nomenclature has generally beenderived using the commercially-available AutoNom software (MDL, SanLeandro, Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of thisinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of this invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from this invention unless specifically indicated.

The values for a and b are independently 0, 1, 2, 3, 4 or 5;particularly independently 0, 1 or 2, and even more particularly 0 or 1.In one embodiment, both a and b are 0.

When present, each R¹ and R² may be at the 2, 3, 4, 5 or 6-position ofthe phenyl ring to which it is attached. Each R¹ and R² areindependently selected from (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl,(3-6C)cycloalkyl, cyano, halo, —OR^(a), —SR^(a), —NR^(a)R^(b),—S(O)R^(c) and —S(O)₂R^(c). Each R^(a) and R^(b) independentlyrepresents hydrogen, (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl or(3-6C)cycloalkyl. Each R^(c) independently represents (1-4C)alkyl,(2-4C)alkenyl, (2-4C)alkynyl or (3-6C)cycloalkyl. Alternatively, twoadjacent R¹ groups or two adjacent R² groups may be joined together toform (3-6C)alkylene, (2-4C)alkylene-O— or —O-(2-4C)alkylene-O—. Inadditions each alkyl group in R¹, R², and R^(a-c) is optionallysubstituted with from 1 to 5 fluoro substituents, and in one embodimentoptionally substituted with 1 to 3 fluoro substituents. In a specificembodiment, R¹ or R² are independently selected from (1-4C)alkyl,fluoro, chloro and —OR^(a). In another specific embodiment, each R¹ andR² is (1-2C)alkyl or fluoro. Representative R¹ and R² groups include,but are not limited to, methyl, ethyl, n-propyl, isopropyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, fluoro, chloro,methoxy, ethoxy, difluoromethoxy and trifluoromethoxy.

The value for c is 0, 1, 2, or 3; particularly 0, 1 or 2; and even moreparticularly 0 or 1. A particular value for c is 0. Particular mentionis made of compounds in which each of a, b and c represents 0.

When present, each R³ independently is fluoro or (1-4C)alkyl. Inaddition, each alkyl group in R³ is optionally substituted with from 1to 5 fluoro substituents, and in one embodiment optionally substitutedwith 1 to 3 fluoro substituents. In a specific embodiment, each R³ isindependently selected from (1-2C)alkyl and fluoro. When two R³substituents are present (c=2), they can be on the same or differentcarbon atoms. Representative R³ groups include, but are not limited to,methyl, ethyl, difluoromethyl, trifluoromethyl and fluoro.

R^(4a) and R^(4b) are independently selected from hydrogen, (1-4C)alkyl,and phenyl-(1-4C)alkyl. R^(4a) and R^(4b) together with the carbon atomto which they are attached may form a (3-6C)heterocyclic ring optionallycontaining one additional heteroatom selected from nitrogen, oxygen orsulfur. This heterocyclic ring is unsubstituted or substituted with 1 or2 substituents selected independently from (1-4C)alkyl and fluoro. Inaddition, each alkyl group in R^(4a) and R^(4b) is optionallysubstituted with from 1 to 5 fluoro substituents, and in one embodimentoptionally substituted with 1 to 3 fluoro substituents. In oneembodiment, R^(4a) and R^(4b) are independently hydrogen or (1-4C)alkyl.In another embodiment R^(4a) and R^(4b) are independently hydrogen or(1-2C)alkyl, such as methyl and ethyl. In yet another embodiment, R^(4a)and R^(4b) are both hydrogen. Alternatively, in another specificembodiment, R^(4a) and R^(4b) are joined together with the nitrogen atomto which they are attached to form a (3-5C)heterocyclic ring optionallycontaining one additional heteroatom selected from nitrogen, oxygen orsulfur. Representative heterocyclic rings include, but are not limitedto, pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl,4-(1-4C)alkylpiperazin-1-yl, morpholin-4-yl and thiomorpholin-4-yl.

The value for e is 1 or 2. In one embodiment, e is 1.

The value for m is 4, 5 or 6. In one embodiment, m is 5.

R⁵ represents hydrogen, (1-4C)alkyl, or (3-4C)cycloalkyl. Examples of(1-4C)alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl and tert-butyl. Examples of (3-4C)cycloalkyl groupsinclude cyclopropyl and cyclobutyl. In addition, each alkyl group in R⁵is optionally substituted with from 1 to 5 fluoro substituents, and inone embodiment optionally substituted with 1 to 3 fluoro substituents.In one embodiment, R⁵ is hydrogen or (1-3C)alkyl, such as methyl, ethyl,n-propyl and isopropyl. In a particular embodiment, R⁵ is hydrogen ormethyl. In another embodiment, R⁵ is hydrogen.

R⁶ is hydrogen or an unbranched (1-4C)alkyl. In addition, each alkylgroup in R⁶ is optionally substituted with from 1 to 5 fluorosubstituents, and in one embodiment optionally substituted with 1 to 3fluoro substituents. Particular values for R⁶ include hydrogen andmethyl. In a particular embodiment, R⁶ is hydrogen. Alternatively, R⁶and R⁸ may be joined, together with the atoms to which they areattached, to form a pyrrolidin-2-yl group. For example, —CHR⁶—NR⁷R⁸forms a pyrrolidin-2-yl group.

R⁷ is selected from hydrogen, (1-6C)alkyl, (3-6C)cycloalkyl, —CH₂Ar¹,—CH₂CH₂—OH and —CH₂CH₂—O-(1-4C)alkyl. In addition, each alkyl group inR⁷ is optionally substituted with from 1 to 5 fluoro substituents, andin one embodiment optionally substituted with 1 to 3 fluorosubstituents. In one embodiment, R⁷ represents hydrogen. In anotherembodiment, R⁷ represents (1-4C)alkyl, including methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.Particular values for R⁷ in this embodiment are methyl, ethyl, n-propyland isopropyl. In a particular embodiment, R⁷ is methyl. In yet anotherembodiment, R⁷ is (3-5C)cycloalkyl, such as cyclopropyl, cyclobutyl andcyclopentyl. In still another embodiment, R⁷ is —CH₂Ar¹, including—CH₂-(phenyl), i.e. benzyl. In yet another embodiment, R⁷ is —CH₂CH₂—OHor —CH₂CH₂—O(1-3C)alkyl, including —CH₂CH₂—O-CH₃, —CH₂CH₂—O—CH₂CH₃,—CH₂CH₂—O—(CH₂)₂CH₃ and —CH₂CH₂—O—CH(CH₃)₂. Particular values for R⁷ inthis embodiment are —CH₂CH₂—OH and —CH₂CH₂—O—CH₃. In another particularembodiment, R⁷ is hydrogen, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, cyclobutyl, cyclopentyl, benzyl, 2-hydroxyethyl or2-methoxyethyl.

R⁸ is hydrogen or (1-6C)alkyl. In addition, each alkyl group in R⁸ isoptionally substituted with from 1 to 5 fluoro substituents, and in oneembodiment optionally substituted with 1 to 3 fluoro substituents. Inone embodiment, R⁸ is hydrogen or (1-4C)alkyl, including methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.Particular values for R⁸ are hydrogen, methyl, ethyl, n-propyl andisopropyl. In a particular embodiment, R⁸ is hydrogen or methyl. Aparticular value for R⁸ is hydrogen. Alternatively, R⁷ and R⁸ togetherwith the nitrogen atom to which they are attached may form apyrrolidin-1-yl, piperidin-1-yl, morpholin-1-yl or thiomorpholin-1-ylgroup. In one embodiment, R⁷ and R⁸ are joined, together with thenitrogen atom to which they are attached, to form a pyrrolidin-1-yl orpiperidin-1-yl group.

Ar¹ represents phenyl or (3-5C)heteroaryl. The phenyl or heteroarylgroup is unsubstituted or substituted with from 1 to 3 substituentsselected independently from halo, (1-4C)alkyl and (1-4C)alkoxy, whereeach alkyl and alkoxy is optionally substituted with from 1 to 3 fluorosubstituents;

In one embodiment, Ar¹ is phenyl, wherein the phenyl group isunsubstituted or substituted with 1, 2 or 3 substituents selectedindependently from halo, (1-4C)alkyl or (1-4C)alkoxy, wherein each alkyland alkoxy group is optionally substituted with from 1 to 3 fluorosubstituents. Representative substituents include fluoro, chloro,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, methoxy, ethoxy, isopropoxy, difluoromethyl,trifluoromethyl, 2,2,2-trifluoroethyl and trifluoromethoxy. A particularvalue for Ar¹ in this embodiment is phenyl.

In another embodiment, Ar¹ is a (3-5C)heteroaryl group containing 1 or 2heteroatoms selected independently from oxygen, nitrogen or sulfur;wherein the heteroaryl group is unsubstituted or substituted with 1 or 2substituents selected independently from halo, (1-4C)alkyl or(1-4C)alkoxy; wherein each alkyl and alkoxy group is optionallysubstituted with from 1 to 3 fluoro substituents. Representativeheteroaryl groups include monovalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, pyrazole, isoxazole, isothiazole,pyridine, pyrazine, pyridazine and pyrimidine, where the point ofattachment is at any available carbon or nitrogen ring atom.Representative substituents include fluoro, chloro, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy,ethoxy, isopropoxy, difluoromethyl, trifluoromethyl,2,2,2-trifluoroethyl and trifluoromethoxy. Particular examples of Ar¹groups in this embodiment include 2-furyl, 2-thienyl and 2-pyridyl.

A particular group of compounds of interest are compound of formula Iwherein R⁵ is hydrogen or (1-4C)alkyl.

A particular group of compounds of interest are compounds of formula Iwherein a and b are 0. Another group of compounds of interest arecompounds of formula I wherein a, b and c are 0. A particular group ofcompounds of interest are compounds of formula I wherein a, b and c are0; and R^(4a) and R^(4b) are hydrogen.

Another particular group of compounds of interest are compounds offormula I wherein a, b and c are 0; R^(4a) and R^(4b) are hydrogen; andR⁵ is hydrogen.

Another particular group of compounds of interest are compounds offormula I wherein a, b and c are 0; R^(4a) and R^(4b) are hydrogen; R⁵is hydrogen; and R⁶ is hydrogen or an unbranched (1-4C)alkyl such asmethyl.

Another particular group of compounds of interest are compounds offormula I, where a, b, and c are 0; R^(4a) and R^(4b) are hydrogen; e is1; m is 5; and R⁵ is hydrogen. These compounds have the formula Ia:

where R⁶, R⁷ and R⁸ are as defined herein; or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof.

Another particular group of compounds of interest are compounds offormula I, where a, b, and c are 0; R^(4a) and R^(4b) are hydrogen; e is1; m is 5; R⁵ is hydrogen; and R⁶ is hydrogen. These compounds have theformula Ib:

where R⁷ and R⁸ are as defined herein; or a pharmaceutically acceptablesalt or solvate or stereoisomer thereof.

Particular compounds of interest include:

-   -   2-{(S)-1-[5-(2-methylamino)acetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;    -   2-{(S)-1-[5-(2-aminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;    -   2-amino-N-{5-[(S)-3-(carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentyl}propionamide;    -   pyrrolidine-2-carboxylic acid        {5-[(S)-3-(carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentyl}amide;    -   N-{5-[(S)-3-(carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentyl}-2-methylaminopropionamide;    -   2-{(S)-1-[5-(2-ethylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;    -   2,2-diphenyl-2-{(S)-1-[5-(2-propylaminoacetylamino)pentyl]pyrrolidin-3-yl}acetamide;    -   2-((S)-1-{5-[2-(2-methoxyethylamino)acetylamino]pentyl}pyrrolidin-3-yl)-2,2-diphenylacetamide;    -   2-{(S)-1-[5-(2-benzylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;    -   2-{(S)-1-[5-(2-dimethylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;    -   2-{(S)-1-[5-(2-cyclopropylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;    -   2-{(S)-1-[5-(2-cyclobutylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;        and    -   2-{(S)-1-[5-(2-cyclopentylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;    -   or a pharmaceutically acceptable salt or solvate thereof.        Definitions

When describing the compounds, compositions, methods and processes ofthis invention, the following terms have the following meanings unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

The term “alkylene” means a divalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like.

The term “alkoxy” means a monovalent group of the formula (alkyl)-O—,where alkyl is as defined herein. Representative alkoxy groups include,by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,sec-butoxy, isobutoxy, tert-butoxy and the like.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to 10 carbonatoms. Representative alkenyl groups include, by way of example,ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and thelike. The term “alkenylene” means a divalent alkenyl group.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbonatoms. Representative alkynyl groups include, by way of example,ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like. The term“alkynylene” means a divalent alkynyl group.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or fused rings (i.e., naphthalene). Unless otherwisedefined, such aryl groups typically contain from 6 to 10 carbon ringatoms. Representative aryl groups include, by way of example, phenyl andnaphthalene-1-yl, naphthalene-2-yl, and the like. The term “arylene”means a divalent aryl group.

The term “azacycloalkyl” means a monovalent heterocyclic ring containingone nitrogen atom, i.e., a cycloalkyl group in which one carbon atom hasbeen replaced with a nitrogen atom. Unless otherwise defined, suchazacycloalkyl groups typically contain from 2 to 9 carbon atoms.Representative examples of an azacycloalkyl group are pyrrolidinyl andpiperidinyl groups. The term “azacycloalkylene” means a divalentazacycloakyl group. Representative examples of an azacycloalkylene groupare pyrrolidinylene and piperidinylene groups.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms. Representative cycloalkylgroups include, by way of example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and the like. The term “cycloalkylene” means a divalentcycloalkyl group.

The term “halo” means fluoro, chloro, bromo and iodo. Examples ofparticular values for halo are fluoro, chloro and bromo.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms. Representative heteroaryl groupsinclude, by way of example, monovalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole,isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine,indole, benzofuran, benzothiophene, benzimidazole, benzthiazole,quinoline, isoquinoline, quinazoline, quinoxaline and the like, wherethe point of attachment is at any available carbon or nitrogen ringatom. The term “heteroarylene” means a divalent heteroaryl group.

The term “heterocyclyl” or “heterocyclic” means a monovalent saturatedor unsaturated (non-aromatic) group having a single ring or multiplecondensed rings and containing in the ring at least one heteroatom(typically 1 to 3 heteroatoms) selected from nitrogen, oxygen or sulfur.Unless otherwise defined, such heterocyclic groups typically containfrom 2 to 9 total ring carbon atoms. Representative heterocyclic groupsinclude, by way of example, monovalent species of pyrrolidine,imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine,thiomorpholine, piperazine, 3-pyrroline and the like, where the point ofattachment is at any available carbon or nitrogen ring atom. The term“heterocyclene” means a divalent heterocyclyl or heterocyclic group.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown in parenthesespreceding the term. For example, the term “(1-4C)alkyl” means an alkylgroup having from 1 to 4 carbon atoms.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Such salts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. Salts derived from pharmaceutically acceptable inorganic basesinclude ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,manganic, manganous, potassium, sodium, zinc and the like. Particularlypreferred are ammonium, calcium, magnesium, potassium and sodium salts.Salts derived from pharmaceutically acceptable organic bases includesalts of primary, secondary and tertiary amines, including substitutedamines, cyclic amines, naturally-occurring amines and the like, such asarginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable acids include acetic, ascorbic,benzenesulfonic, benzoic, camphosulfonic, citric, ethanesulfonic,edisylic, fumaric, gentisic, gluconic, glucoronic, glutamic, hippuric,hydrobromic, hydrochloric, isethionic, lactic, lactobionic, maleic,malic, mandelic, methanesulfonic, mucic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic, nicotinic,nitric, orotic, pamoic, pantothenic, phosphoric, succinic, sulfuric,tartaric, p-toluenesulfonic, xinafoic and the like. Particularlypreferred are citric, hydrobromic, hydrochloric, isethionic, maleic,naphthalene-1,5-disulfonic, phosphoric, sulfuric and tartaric acids.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. Preferably, the salt is a pharmaceuticallyacceptable salt, although this is not required for salts of intermediatecompounds that are not intended for administration to a patient.

The term “solvate” as used herein refers to a complex or aggregateformed by one or more molecules of a solute, i.e. a compound of theinvention or a pharmaceutically-acceptable salt thereof, and one or moremolecules of a solvent. Such solvates are typically crystalline solidshaving a substantially fixed molar ratio of solute and solvent.Representative solvents include by way of example, water, methanol,ethanol, isopropanol, acetic acid, and the like. When the solvent iswater, the solvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof” is intended to include allpermutations of salts, solvates and stereoisomers, such as a solvate ofa pharmaceutically acceptable salt of a stereoisomer of a compound offormula (I).

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a patient,such as a mammal (particularly a human) that includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; sulfonic ester groups, such as mesylate, tosylate, brosylate,nosylate and the like; and acyloxy groups, such as acetoxy,trifluoroacetoxy and the like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected from undesired reactions with a protecting orblocking group. Functional groups which may be protected include, by wayof example, carboxylic acid groups, amino groups, hydroxyl groups, thiolgroups, carbonyl groups and the like. Representative protecting groupsfor carboxylic acids include esters (such as a p-methoxybenzyl ester),amides and hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, N.Y., 1999, and references cited therein.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,1,1-di-(4′-methoxyphenyl)methyl; tert-butoxycarbonyl (BOC), benzyl (Bn),trityl (Tr), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc),formyl, trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), and thelike.

The term “carboxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a carboxy group. Representativecarboxy-protecting groups include, but are not limited to, esters, suchas methyl, ethyl, tert-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluroenylmethyl (Fm), trimethylsilyl (TMS), tert-butyldimethylsilyl(TBS), diphenylmethyl (benzhydryl, DPM) and the like.

The term “hydroxyl-protecting group” means a protecting group suitablefor preventing undesirable reactions at a hydroxyl group. Representativehydroxyl-protecting groups include, but are not limited to, silyl groupsincluding tri(1-6C)alkylsilyl groups, such as trimethylsilyl (TMS),triethylsilyl (TES), tert-butyldimethylsilyl (TBS) and the like; esters(acyl groups) including (1-6C)alkanoyl groups, such as formyl, acetyland the like; arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl(PMB), 9-fluorenylmethyl (Fm), diphenylmethyl (benzhydryl, DPM) and thelike. Additionally, two hydroxyl groups can also be protected as analkylidene group, such as prop-2-ylidine, formed, for example, byreaction with a ketone, such as acetone.

General Synthetic Procedures

The diphenylmethyl compounds of this invention can be prepared fromreadily available starting materials using the following general methodsand procedures or by using other information readily available to thoseof ordinary skill in the art. Although a particular embodiment of thepresent invention may be shown or described herein, those skilled in theart will recognize that all embodiments or aspects of the presentinvention can be prepared using the methods described herein or by usingother methods, reagents and starting materials known to those skilled inthe art. It will also be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. While the optimumreaction conditions may vary depending on the particular reactants orsolvent used, such conditions can be readily determined by one skilledin the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions for protection and deprotection of suchfunctional groups are well-known in the art. Protecting groups otherthan those illustrated in the procedures described herein may be used,if desired. For example, numerous protecting groups, and theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, N.Y.,1999, and references cited therein.

By way of illustration, the compounds of formula I can be prepared by aprocess comprising:

-   -   (a) reacting a compound of formula II:        with a compound of formula III:        X¹—(CH₂)_(m)—NR⁵CO—CHR⁶—NR⁷R⁸   III        wherein X¹ represents a leaving group;    -   (b) coupling a compound of formula IV:        with a compound of formula V:        HOOC—CHR⁶—NR⁷R⁸   V        or a reactive derivative thereof;    -   (c) reacting a compound of formula II with a compound of formula        VI:        OHC—(CH₂)_(m-1)—NR⁵CO—CHR⁶—NR⁷R⁸   VI        in the presence of a reducing agent; or    -   (d) for a compound of formula (I) in which R⁶ represents a        hydrogen atom or an unbranched (1-4C)alkyl group, reacting a        compound of formula VII:        wherein X² represents a leaving group, with a compound of        formula VIII:        HNR⁷R⁸   VIII        to provide a compound of formula I; and optionally, forming a        pharmaceutically acceptable salt thereof.

In these reactions, depending upon the particular substituents present,one or more protecting group may be employed. If such protecting groupsare used, they are removed using conventional procedures to provide thecompound of formula I.

Generally, if a salt of one of the starting materials is used in theprocesses described above, such as an acid addition salt, the salt istypically neutralized before or during the reaction process. Thisneutralization reaction is typically accomplished by contacting the saltwith one molar equivalent of a base for each molar equivalent of acidaddition salt.

In process (a), the reaction between the compounds of formula II andIII, the leaving group represented by X¹ can be, for example, halo, suchas chloro, bromo, or iodo; or a sulfonic acid ester group, such asmesylate or tosylate. The reaction is conveniently performed in thepresence of a base, for example a tertiary amine such asdiisopropylethylamine. Convenient solvents include nitrites, such asacetonitrile. The reaction is conveniently conducted at a temperature inthe range of from 0° C. to 100° C.

Compounds of formula II may be prepared as described in U.S. Pat. No.5,096,890 to Cross et al., the disclosure of which is incorporatedherein by reference in its entirety.

Alternatively, compounds of formula II can be prepared by deprotecting acompound of formula IX:

in which P¹ represents an amino-protecting group, such as a benzylgroup. Benzyl groups are conveniently removed by reduction, for example,using a hydrogen or ammonium formate and a Group VIII metal catalyst,such as palladium.

Compounds of formula IX can be prepared by reacting a carboxylic acid offormula X:

with an amine of formula HNR^(4a)R^(4b) under amide bond formingconditions.

Compounds of formula X may be prepared by hydrolyzing a compound offormula XI:

Compounds of formula XI can be prepared as described in U.S Pat. No.5,096,890 to Cross et al..

Compounds of formula III can be prepared from a compounds of formulaXII:HO—(CH₂)_(m)—NR⁵NR⁵CO—CHR⁶—NR⁷R⁸   XIIby, for example, reacting compound XII with a halogenating agent, suchas thionyl chloride, or with an appropriate sulfonyl chloride, such asp-toluenesulfonyl chloride.

Compounds of formula XII can be prepared from an amino alcohol and anamino acid using conventional coupling reagents and reaction conditions.For example, representative conditions are described in Example 1herein.

In process (b), the coupling reaction can be conducted using anyconventional amide coupling reagents and reaction conditions. Forexample, a carboxylic acid halide, such as a carboxylic acid chloride,of compound V can be used or a dehydrating agent, such as HATU(O-(7-azabenzo-triazol-1-yl-N,N,N′,N′-tetramethyluronium-hexafluorophosphate)may be employed in this reaction to activate the carboxylic acidtypically in the presence of a tertiary amine, such asdiisopropylethylamine. Convenient solvents include amides, such asdimethylformamide. The coupling is conveniently performed at atemperature in the range of from 0° C. to 100° C.

Compounds of formula IV can be prepared by reacting a compound offormula XIII:

with an amine of formula XIV:H₂NR⁵   XIVin the presence of a reducing agent as described, for example, inprocess (c) herein.

Compounds of formula XIII can be prepared by reacting a compound offormula XV:

with a suitable oxidizing agent, such as sulfur trioxide pyridinecomplex in dimethyl sulfoxide in the presence of a tertiary amine base,such as diisopropylethylamine.

Compounds of formula XV can be prepared by reacting a compound offormula II with a compound of formula XVI:X³—(CH₂)_(m)—OH   XVIin which X³ represents a leaving group including, for example, halo,such as chloro, bromo, or iodo; or a sulfonic acid ester group, such asmesylate or tosylate. Representative conditions for this reaction aredescribed, for example, in process step (a) and Example 2 herein.

Alternatively, compounds of formula IV can be prepared by reacting acompound of formula II with a compound of formula XVII:X³—(CH₂)_(m)—NP²P³   XVIIin which P² represents an amino-protecting group (such astert-butoxycarbonyl) and P³ represents R⁵ or an amino-protecting group(such as tert-butoxycarbonyl), followed by the removal of theamino-protecting group or groups. In a variation of this process, p² andP³ are joined, together with the nitrogen atom to which they areattached, to form a phthalimido group. After reaction with compound II,such a phthalimido group may be removed with hydrazine to afford acompound of formula IV in which R⁵ represents hydrogen.

In process (c), the reducing agent can be, for example, hydrogen in thepresence of a Group VIII metal catalyst, such as palladium, or a metalhydride reducing agent, including a borohydride, such as sodiumtriacetoxyborohydride. This reaction is conveniently performed at atemperature in the range of from 0° C. to 100° C. Convenient solventsinclude halogenated hydrocarbons, such as dichloroethane and alcohols,such as methanol, or combinations thereof.

Compounds of formula VI can be prepared by oxidizing a compound offormula XVIII:HO—(CH₂)_(m)—NR⁵CO—CHR⁶—NR⁷R⁸   XVIIIusing a suitable oxidizing agent, such as sulfur trioxide pyridinecomplex in dimethyl sulfoxide in the presence of a tertiary amine base,such as diisopropylethylamine.

In process (d), the leaving group represented by X² may be, for example,halo, such as chloro, bromo, or iodo; or a sulfonic acid ester group,such as mesylate or tosylate. When X² is chloro, the reaction isconveniently conducted in the presence of an alkali metal iodide, suchas sodium iodide. Typically, this reaction is conducted at a temperaturein the range of from 0° C. to 120° C. Convenient solvents includeamides, such as dimethylformamide.

Compounds of formula VII may be prepared from a compound of formula IVand an appropriate carboxylic acid or reactive derivative thereof, suchas chloroacetyl chloride. Conditions for this reaction are illustrated,for example in Example 6 herein.

It will be appreciated that certain compounds of formula I, for example,those in which R⁷ represents —(CH₂)Ar¹, such as benzyl, may themselvesserve as intermediates for other compounds of formula I. Thus, a—(CH₂)Ar¹ group may serve as a protecting group and may be removed bycatalytic hydrogenation, for example, in the presence of a catalyst,such as palladium.

Certain of the intermediates described herein are believed to be noveland accordingly, such compounds are provided as further aspects of theinvention including, for example, the compounds of formula V, VI and VIIand salts thereof.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of this invention orintermediates thereof are described in the Examples set forth below.

Pharmaceutical Compositions and Formulations

The diphenylmethyl compounds of this invention are typicallyadministered to a patient in the form of a pharmaceutical composition orformulation. Such pharmaceutical compositions may be administered to thepatient by any acceptable route of administration including, but notlimited to, inhaled, oral, nasal, topical (including transdermal) andparenteral modes of administration.

It will be understood that any form of the compounds of this invention,(i.e., free base, pharmaceutically acceptable salt, solvate, etc.) thatis suitable for the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one of its composition aspects, this invention isdirected to a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or excipient and a therapeutically effective amountof a compound of formula I, or a pharmaceutically acceptable salt orsolvate or stereoisomer thereof. Optionally, such pharmaceuticalcompositions may contain other therapeutic and/or formulating agents ifdesired.

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of a compound of the present inventionor a pharmaceutically acceptable salt or solvate or stereoisomerthereof. Typically, such pharmaceutical compositions will contain fromabout 0.01 to about 95% by weight of the active agent; including, fromabout 0.01 to about 30% by weight; such as from about 0.01 to about 10%by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of this invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;(21) compressed propellant gases, such as chlorofluorocarbons andhydrofluorocarbons; and (22) other non-toxic compatible substancesemployed in pharmaceutical compositions.

The pharmaceutical compositions of this invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills, canisters, cartridges, dispensers and the like using conventionalprocedures and equipment.

In one embodiment, the pharmaceutical compositions of this invention aresuitable for inhaled administration. Suitable pharmaceuticalcompositions for inhaled administration will typically be in the form ofan aerosol or a powder. Such compositions are generally administeredusing well-known delivery devices, such as a nebulizer inhaler, ametered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similardelivery device.

In a specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a nebulizer inhaler. Such nebulizer devices typically produce astream of high velocity air that causes the pharmaceutical compositioncomprising the active agent to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Alternatively, the active agent can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where micronized is typicallydefined as having about 90% or more of the particles with a diameter ofless than about 10 μm. Suitable nebulizer devices are providedcommercially, for example, by PARI GmbH (Stamberg, German). Othernebulizer devices include Respimat (Boehringer Ingelheim) and thosedisclosed, for example, in U.S. Pat. No. 6,123,068 to Lloyd et al. andWO 97/12687 (Eicher et al.).

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an isotonic aqueous solution comprising from about0.05 μg/mL to about 10 mg/mL of a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

In another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a dry powder inhaler. Such dry powder inhalers typicallyadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. In order to achieve a freeflowing powder, the active agent is typically formulated with a suitableexcipient such as lactose or starch.

A representative pharmaceutical composition for use in a dry powderinhaler comprises dry lactose having a particle size between about 1 μmand about 100 μm and micronized particles of a compound of formula I, ora pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Such a dry powder formulation can be made, for example, by combining thelactose with the active agent and then dry blending the components.Alternatively, if desired, the active agent can be formulated without anexcipient. The pharmaceutical composition is then typically loaded intoa dry powder dispenser, or into inhalation cartridges or capsules foruse with a dry powder delivery device.

Examples of dry powder inhaler delivery devices include Diskhaler(GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g., U.S. Pat.No. 5,035,237 to Newell et al.); Diskus (GlaxoSmithKline) (see, e.g.,U.S. Pat. No. 6,378,519 to Davies et al.); Turbuhaler (AstraZeneca,Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769 to Wetterlin);Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 4,353,365 toHallworth et al.) and Handihaler (Boehringer Ingelheim). Furtherexamples of suitable DPI devices are described in U.S. Pat. No.5,415,162 to Casper et al., U.S. Pat. No. 5,239,993 to Evans, and U.S.Pat. No. 5,715,810 to Armstrong et al., and references cited therein.

In yet another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a metered-dose inhaler. Such metered-dose inhalers typicallydischarge a measured amount of the active agent or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof using compressedpropellant gas. Accordingly, pharmaceutical compositions administeredusing a metered-dose inhaler typically comprise a solution or suspensionof the active agent in a liquefied propellant. Any suitable liquefiedpropellant may be employed including chlorofluorocarbons, such as CCl₃F,and hydrofluoroalkanes (HFAs), such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). Due toconcerns about chlorofluorocarbons affecting the ozone layer,formulations containing HFAs are generally preferred. Additionaloptional components of HFA formulations include co-solvents, such asethanol or pentane, and surfactants, such as sorbitan trioleate, oleicacid, lecithin, and glycerin. See, for example, U.S. Pat. No. 5,225,183to Purewal et al., EP 0717987 A2 (Minnesota Mining and ManufacturingCompany), and WO 92/22286 (Minnesota Mining and Manufacturing Company).

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of a compoundof formula I, or a pharmaceutically acceptable salt or solvate orstereoisomer thereof; from about 0% to about 20% by weight ethanol; andfrom about 0% to about 5% by weight surfactant; with the remainder beingan HFA propellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing theactive agent, ethanol (if present) and the surfactant (if present). Toprepare a suspension, the active agent is micronized and then combinedwith the propellant. The formulation is then loaded into an aerosolcanister, which forms a portion of a metered-dose inhaler device.Examples of metered-dose inhaler devices developed specifically for usewith HFA propellants are provided in U.S. Pat. No. 6,006,745 to Mareckiand U.S. Pat. No. 6,143,277 to Ashurst et al. Alternatively, asuspension formulation can be prepared by spray drying a coating ofsurfactant on micronized particles of the active agent. See, forexample, WO 99/53901 (Glaxo Group Ltd.) and WO 00/61108 (Glaxo GroupLtd.).

For additional examples of processes of preparing respirable particles,and formulations and devices suitable for inhalation dosing see U.S.Patent No. 6,268,533 to Gao et al., U.S. Pat. No. 5,983,956 to Trofast,U.S. Pat. No. 5,874,063 to Briggner et al., and U.S. Pat. No. 6,221,398to Jakupovic et al.; and WO 99/55319 (Glaxo Group Ltd.) and WO 00/30614(AstraZeneca AB).

In another embodiment, the pharmaceutical compositions of this inventionare suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof this invention will typically comprise a compound of the presentinvention as the active ingredient and one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate.Optionally or alternatively, such solid dosage forms may also comprise:(1) fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and/or glycerol monostearate; (8)absorbents, such as kaolin and/or bentonite clay; (9) lubricants, suchas talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and/or mixtures thereof; (10) coloringagents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of this invention. Examples ofpharmaceutically acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PVAP), hydroxypropyl methylcellulose phthalate,methacrylic-methacrylic acid ester copolymers, cellulose acetatetrimellitate (CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropylmethyl cellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (e.g., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof this invention are preferably packaged in a unit dosage form. Theterm “unit dosage form” means a physically discrete unit suitable fordosing a patient, i.e., each unit containing a predetermined quantity ofactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

The compounds of this invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example, acompound of this invention can be admixed with permeation enhancers,such as propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The pharmaceutical compositions of this invention may also contain othertherapeutic agents that are co-administered with a compound of formulaI, or pharmaceutically acceptable salt or solvate or stereoisomerthereof. For example, the pharmaceutical compositions of this inventionmay further comprise one or more therapeutic agents selected from otherbronchodilators (e.g., PDE₃ inhibitors, adenosine 2b modulators and β₂adrenergic receptor agonists); anti-inflammatory agents (e.g., steroidalanti-inflammatory agents, such as corticosteroids; non-steroidalanti-inflarnmatory agents (NSAIDs), and PDE₄ inhibitors); othermuscarinic receptor antagonists (i.e., antichlolinergic agents);antiinfective agents (e.g., Gram positive and Gram negative antibioticsor antivirals); antihistamines; protease inhibitors; and afferentblockers (e.g., D₂ agonists and neurokinin modulators). In oneparticular aspect of the invention, the compound of the invention isco-administered with a β₂ adrenergic receptor agonist and a steroidalanti-inflammatory agent. The other therapeutic agents can be used in theform of pharmaceutically acceptable salts or solvates. Additionally, ifappropriate, the other therapeutic agents can be used as optically purestereoisomers.

Representative β₂ adrenergic receptor agonists that can be used incombination with the compounds of this invention include, but are notlimited to, salmeterol, salbutamol, formoterol, salmefamol, fenoterol,terbutaline, albuterol, isoetharine, metaproterenol, bitolterol,pirbuterol, levalbuterol and the like, or pharmaceutically acceptablesalts thereof. Other β₂ adrenergic receptor agonists that can be used incombination with the compounds of this invention include, but are notlimited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}-amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds disclosed in WO 02/066422 (Glaxo Group Ltd.);3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-phenyl]imidazolidine-2,4-dioneand related compounds disclosed in WO 02/070490 (Glaxo Group Ltd.);3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide and related compounds disclosed inWO 02/076933 (Glaxo Group Ltd.);4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds disclosed in WO 03/024439 (Glaxo Group Ltd.);N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,576,793 to Moran etal.;N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,653,323 to Moran etal.; and pharmaceutically acceptable salts thereof. In a particularembodiment, the β₂-adrenoreceptor agonist is a crystallinemonohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine. When employed, the β₂-adrenoreceptor agonist will be presentin the pharmaceutical composition in a therapeutically effective amount.Typically, the β₂-adrenoreceptor agonist will be present in an amountsufficient to provide from about 0.05 μg to about 500 μg per dose.

Representative steroidal anti-inflammatory agents that can be used incombination with the compounds of this invention include, but are notlimited to, methyl prednisolone, prednisolone, dexamethasone,fluticasone propionate,6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydrofuran-3S-yl) ester, beclomethasone esters (e.g.,the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g., the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,ST-1126 and the like, or pharmaceutically-acceptable salts thereof. Whenemployed, the steroidal anti-inflammatory agent will be present in thepharmaceutical composition in a therapeutically effective amount.Typically, the steroidal anti-inflammatory agent will be present in anamount sufficient to provide from about 0.05 μg to about 500 μg perdose.

An exemplary combination is a compound of formula I, or pharmaceuticallyacceptable salt or solvate or stereoisomer thereof, co-administered withsalmeterol as the β₂ adrenergic receptor agonist, and fluticasonepropionate as the steroidal anti-inflammatory agent. Another exemplarycombination is a compound of formula I, or pharmaceutically acceptablesalt or solvate or stereoisomer thereof, co-administered with acrystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineas the β₂-adrenoreceptor agonist, and6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester as the steroidal anti-inflammatory agent.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (such as sodium cromoglycate;nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g.,theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g., monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors, such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g., adenosine 2a agonists); cytokineantagonists (e.g., chemokine antagonists such as, an interleukinantibody (αIL antibody), specifically, an αIL-4 therapy, an αIL-13therapy, or a combination thereof); or inhibitors of cytokine synthesis.

For example, representative phosphodiesterase-4 (PDE4) inhibitors ormixed PDE3/PDE4 inhibitors that can be used in combination with thecompounds of this invention include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inW099/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vemalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

Representative muscarinic antagonists (i.e., anticholinergic agents)that can be used in combination with, and in addition to, the compoundsof this invention include, but are not limited to, atropine, atropinesulfate, atropine oxide, methylatropine nitrate, homatropinehydrobromide, hyoscyamine (d, l) hydrobromide, scopolamine hydrobromide,ipratropium bromide, oxitropium bromide, tiotropium bromide,methantheline, propantheline bromide, anisotropine methyl bromide,clidinium bromide, copyrrolate (Robinul), isopropamide iodide,mepenzolate bromide, tridihexethyl chloride (Pathilone), hexocycliummethylsulfate, cyclopentolate hydrochloride, tropicamide,trihexyphenidyl hydrochloride, pirenzepine, telenzepine, AF-DX 116 andmethoctramine and the like, or a pharmaceutically acceptable saltthereof; or, for those compounds listed as a salt, alternatepharmaceutically acceptable salt thereof.

Representative antihistamines (i.e., H₁-receptor antagonists) that canbe used in combination with the compounds of this invention include, butare not limited to, ethanolamines, such as carbinoxamine maleate,clemastine fumarate, diphenylhydramine hydrochloride and dimenhydrinate;ethylenediamines, such as pyrilamine amleate, tripelennaminehydrochloride and tripelennamine citrate; alkylamines, such aschlorpheniramine and acrivastine; piperazines, such as hydroxyzinehydrochloride, hydroxyzine pamoate, cyclizine hydrochloride, cyclizinelactate, meclizine hydrochloride and cetirizine hydrochloride;piperidines, such as astemizole, levocabastine hydrochloride, loratadineor its descarboethoxy analogue, terfenadine and fexofenadinehydrochloride; azelastine hydrochloride; and the like, or apharmaceutically acceptable salt thereof; or, for those compounds listedas a salt, alternate pharmaceutically acceptable salt thereof.

Suitable doses for the other therapeutic agents administered incombination with a compound of the invention are in the range of about0.05 μg/day to about 100 mg/day.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

FORMULATION EXAMPLE A

A dry powder for administration by inhalation is prepared as follows:Ingredients Amount Compound of the invention 0.2 mg Lactose  25 mg

Representative Procedure: The compound of the invention is micronizedand then blended with lactose. This blended mixture is then loaded intoa gelatin inhalation cartridge. The contents of the cartridge areadministered using a powder inhaler.

FORMULATION EXAMPLE B

A dry powder formulation for use in a dry powder inhalation device isprepared as follows:

Representative Procedure: A pharmaceutical composition is preparedhaving a bulk formulation ratio of micronized compound of the inventionto lactose of 1:200. The composition is packed into a dry powderinhalation device capable of delivering between about 10 μg and about100 μg of the compound of the invention per dose.

FORMULATION EXAMPLE C

A dry powder for administration by inhalation in a metered dose inhaleris prepared as follows:

Representative Procedure: A suspension containing 5 wt % of a compoundof the invention and 0.1 wt % lecithin is prepared by dispersing 10 g ofthe compound of the invention as micronized particles with mean sizeless than 10 μm in a solution formed from 0.2 g of lecithin dissolved in200 mL of demineralized water. The suspension is spray dried and theresulting material is micronized to particles having a mean diameterless than 1.5 μm. The particles are loaded into cartridges withpressurized 1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE D

A pharmaceutical composition for use in a metered dose inhaler isprepared as follows:

Representative Procedure: A suspension containing 5 wt % compound of theinvention, 0.5 wt % lecithin, and 0.5 wt % trehalose is prepared bydispersing 5 g of active ingredient as micronized particles with meansize less than 10 μm in a colloidal solution formed from 0.5 g oftrehalose and 0.5 g of lecithin dissolved in 100 mL of demineralizedwater. The suspension is spray dried and the resulting material ismicronized to particles having a mean diameter less than 1.5 μm. Theparticles are loaded into canisters with pressurized1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE E

A pharmaceutical composition for use in a nebulizer inhaler is preparedas follows:

Representative Procedure: An aqueous aerosol formulation for use in anebulizer is prepared by dissolving 0.1 mg of the compound of theinvention in 1 mL of a 0.9% NaCl solution acidified with citric acid.The mixture is stirred and sonicated until the active ingredient isdissolved. The pH of the solution is adjusted to a value in the range offrom 3 to 8 by the slow addition of NaOH.

FORMULATION EXAMPLE F

Hard gelatin capsules for oral administration are prepared as follows:Ingredients Amount Compound of the invention 250 mg Lactose(spray-dried) 200 mg Magnesium stearate  10 mg

Representative Procedure: The ingredients are thoroughly blended andthen loaded into a hard gelatin capsule (460 mg of composition percapsule).

FORMULATION EXAMPLE G

A suspension for oral administration is prepared as follows: IngredientsAmount Compound of the invention 1.0 g Fumaric acid 0.5 g Sodiumchloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 g Granulatedsugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k (Vanderbilt Co.)1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilled water q.s. to 100 mL

Representative Procedure: The ingredients are mixed to form a suspensioncontaining 100 mg of active ingredient per 10 mL of suspension.

FORMULATION EXAMPLE H

An injectable formulation is prepared as follows: Ingredients AmountCompound of the invention 0.2 g Sodium acetate buffer solution (0.4 M)2.0 mL HCl (0.5 N) or NaOH (0.5 N) q.s. to pH 4 Water (distilled,sterile) q.s. to 20 mL

Representative Procedure: The above ingredients are blended and the pHis adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.

Utility

The diphenylmethyl compounds of this invention are expected to be usefulas muscarinic receptor antagonists and therefore, such compounds areexpected to be useful for treating medical conditions mediated bymuscarinic receptors, i.e., medical conditions which are ameliorated bytreatment with a muscarinic receptor antagonist. Such medical conditionsinclude, by way of example, pulmonary disorders or diseases includingthose associated with reversible airway obstruction, such as chronicobstructive pulmonary disease (e.g., chronic and wheezy bronchitis andemphysema), asthma, pulmonary fibrosis, allergic rhinitis, rhinorrhea,and the like. Other medical conditions that can be treated withmuscarinic receptor antagonists are genitourinary tract disorders, suchas overactive bladder or detrusor hyperactivity and their symptoms;gastrointestinal tract disorders, such as irritable bowel syndrome,diverticular disease, achalasia, gastrointestinal hypermotilitydisorders and diarrhea; cardiac arrhythmias, such as sinus bradycardia;Parkinson's disease; cognitive disorders, such as Alzheimer's disease;dismenorrhea; and the like.

In one embodiment, the compounds of this invention are useful fortreating smooth muscle disorders in mammals, including humans and theircompanion animals (e.g., dogs, cats etc.). Such smooth muscle disordersinclude, by way of illustration, overactive bladder, chronic obstructivepulmonary disease and irritable bowel syndrome.

When used to treat smooth muscle disorders or other conditions mediatedby muscarinic receptors, the compounds of this invention will typicallybe administered orally, rectally, parenterally or by inhalation in asingle daily dose or in multiple doses per day. The amount of activeagent administered per dose or the total amount administered per daywill typically be determined by the patient's physician and will dependon such factors as the nature and severity of the patients condition,the condition being treated, the age and general health of the patient,the tolerance of the patient to the active agent, the route ofadministration and the like.

Typically, suitable doses for treating smooth muscle disorders or otherdisorders mediated by muscarinic receptors will range from about 0.14μg/kg/day to about 7 mg/kg/day of active agent; including from about0.15 μg/kg/day to about 5 mg/kg/day. For an average 70 kg human, thiswould amount to about 10 μg per day to about 500 mg per day of activeagent.

In a specific embodiment, the compounds of this invention are useful fortreating pulmonary or respiratory disorders, such as COPD or asthma, inmammals including humans. When used to treat such disorders, thecompounds of this invention will typically be administered by inhalationin multiple doses per day, in a single daily dose or a single weeklydose. Generally, the dose for treating a pulmonary disorder will rangefrom about 10 μg/day to about 200 μg/day. As used herein, COPD includeschronic obstructive bronchitis and emphysema (see, for example, Barnes,Chronic Obstructive Pulmonary Disease, N Engl J Med 343:269-78 (2000)).

When used to treat a pulmonary disorder, the compounds of this inventionare optionally administered in combination with other therapeutic agentssuch as a β₂-adrenoreceptor agonist; a corticosteroid, a non-steroidalanti-inflammatory agent, or combinations thereof.

When administered by inhalation, the compounds of this inventiontypically have the effect of producing bronchodilation. Accordingly, inanother of its method aspects, this invention is directed to a method ofproducing bronchodilation in a patient, the method comprisingadministering to a patient a bronchodilation-producing amount of acompound of the invention. Generally, the therapeutically effective dosefor producing bronchodilation will range from about 10 μg/day to about200 μg/day.

In another embodiment, the compounds of this invention are used to treatoveractive bladder. When used to treat overactive bladder, the compoundsof this invention will typically be administered orally in a singledaily dose or in multiple doses per day; preferably in a single dailydose. Preferably, the dose for treating overactive bladder will rangefrom about 1.0 to about 500 mg/day.

In yet another embodiment, the compounds of this invention are used totreat irritable bowel syndrome. When used to treat irritable bowelsyndrome, the compounds of this invention will typically be administeredorally or rectally in a single daily dose or in multiple doses per day.Preferably, the dose for treating irritable bowel syndrome will rangefrom about 1.0 to about 500 mg/day.

Since compounds of this invention are muscarinic receptor antagonists,such compounds are also useful as research tools for investigating orstudying biological systems or samples having muscarinic receptors. Suchbiological systems or samples may comprise M₁, M₂, M₃, M₄ and/or M₅muscarinic receptors. Any suitable biological system or sample havingmuscarinic receptors may be employed in such studies which may beconducted either in vitro or in vivo. Representative biological systemsor samples suitable for such studies include, but are not limited to,cells, cellular extracts, plasma membranes, tissue samples, mammals(such as mice, rats, guinea pigs, rabbits, dogs, pigs, etc.), and thelike.

In this embodiment, a biological system or sample comprising amuscarinic receptor is contacted with a muscarinic receptor-antagonizingamount of a compound of this invention. The effects of antagonizing themuscarinic receptor are then determined using conventional proceduresand equipment, such as radioligand binding assays and functional assays.Such functional assays include ligand-mediated changes in intracellularcyclic adenosine monophosphate (cAMP), ligand-mediated changes inactivity of the enzyme adenylyl cyclase (which synthesizes cAMP),ligand-mediated changes in incorporation of guanosine5′-O-(γ-thio)triphosphate ([³⁵S]GTPγS) into isolated membranes viareceptor catalyzed exchange of [³⁵S]GTPγS for GDP, ligand-mediatedchanges in free intracellular calcium ions (measured, for example, witha fluorescence-linked imaging plate reader or FLIPR® from MolecularDevices, Inc.). A compound of this invention will antagonize or decreasethe activation of muscarinic receptors in any of the functional assayslisted above, or assays of a similar nature. A muscarinicreceptor-antagonizing amount of a compound of this invention willtypically range from about 0.1 nanomolar to about 100 nanomolar.

Additionally, the compounds of this invention can be used as researchtools for discovering new compounds that have muscarinic receptorantagonist activity. In this embodiment, muscarinic receptor bindingdata (e.g., as determined by in vitro radioligand displacement assays)for a test compound or a group of test compounds is compared to themuscarinic receptor binding data for a compound of this invention toidentify those test compounds that have about equal or superiormuscarinic receptor binding, if any. This aspect of the inventionincludes, as separate embodiments, both the generation of comparisondata (using the appropriate assays) and the analysis of the test data toidentify test compounds of interest.

In another embodiment, the compounds of this invention are used toantagonize a muscarinic receptor in biological system, and a mammal inparticular, such as mice, rats, guinea pigs, rabbits, dogs, pigs, humansand so forth. In this embodiment, a therapeutically effective amount ofthe compound of formula I is administered to the mammal. The effects ofantagonizing the muscarinic receptor can then determined usingconventional procedures and equipment, examples of which are describedabove.

Among other properties, compounds of this invention have been found tobe potent inhibitors of M₃ muscarinic receptor activity. Accordingly, ina specific embodiment, this invention is directed to compounds offormula I having an inhibition dissociation constant (K_(i)) for the M₃receptor subtype of less than or equal to 100 nM; preferably, less thanor equal to 50 nM; and more preferably, less than or equal to 10 nM (asdetermined, for example, by an in vitro radioligand displacement assay).

Additionally, compounds of this invention are expected to possess adesirable duration of action. Accordingly, in another specificembodiment, this invention is directed to compounds of formula I havinga duration of action greater than about 24 hours.

Moreover, compounds of this invention are also expected to possessreduced side effects, such as dry mouth, at efficacious doses whenadministered by inhalation compared to other known muscarinic receptorantagonists administered by inhalation (such as tiotropium).

These properties, as well as the utility of the compounds of thisinvention, can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. For example, representativeassays are described in further detail in the following Examples.

EXAMPLES

The following Preparations and Examples illustrate specific embodimentsof this invention. In these examples, the following abbreviations havethe following meanings:

-   -   AC adenylyl cyclase    -   ACh acetylcholine    -   ACN acetonitrile    -   BOC tert-butoxycarbonyl    -   BSA bovine serum albumin    -   cAMP 3′-5′ cyclic adenosine monophosphate    -   CHO Chinese hamster ovary    -   cM₅ cloned chimpanzee M₅ receptor    -   DABCO 1,4-diazabicyclo[2.2.2]octane    -   DCM dichloromethane (i.e., methylene chloride)    -   DIPEA N,N-diisopropylethylamine    -   dPBS Dulbecco's phosphate buffered saline    -   DMF dimethylformamide    -   EDTA ethylenediamine tetraacetic acid    -   EtOAc ethyl acetate    -   EtOH ethanol    -   FBS fetal bovine serum    -   FLIPR fluorometric imaging plate reader    -   HATU O-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluronium        hexafluorophosphate    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   hM₁ cloned human M₁ receptor    -   hM₂ cloned human M₂ receptor    -   hM₃ cloned human M₃ receptor    -   hM₄ cloned human M₄ receptor    -   hM₅ cloned human M₅ receptor    -   HPLC high-performance liquid chromatography    -   IPAc isopropyl acetate    -   MCh methylcholine    -   MeOH methanol    -   MTBE methyl tert-butyl ether    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, all materials, suchas reagents, starting materials and solvents, were purchased fromcommercial suppliers (such as Sigma-Aldrich, Fluka, and the like) andwere used without further purification.

Unless otherwise indicated, HPLC analysis was conducted using an Agilent(Palo Alto, Calif.) Series 1100 instrument equipped with a Zorbax BonusRP 2.1×50 mm column (Agilent) having a 3.5 micron particle size.Detection was by UV absorbance at 214 nm. The mobile phases employedwere as follows (by volume): A is ACN (2%), water (98%) and TFA (0.1%);and B is ACN (90%), water (10%) and TFA (0.1%). HPLC 10-70 data wasobtained using a flow rate of 0.5 mL/minute of 10 to 70% B over a 6minute gradient (with the remainder being A). Similarly, HPLC 5-35 dataand HPLC 10-90 data were obtained using 5 to 35% B; or 10 to 90% B overa 5 minute gradient.

Liquid chromatography mass spectrometry (LCMS) data were obtained withan Applied Biosystems (Foster City, Calif.) Model API-150EX instrument.LCMS 10-90 data was obtained using 10 to 90% Mobile Phase B over a 5minute gradient.

Small-scale purification was conducted using an API 150EX PrepWorkstation system from Applied Biosystems. The mobile phases employedwere as follows (by volume): A is water and 0.05% TFA; and B is ACN and0.05% TFA. For arrays (typically about 3 to 50 mg recovered sample size)the following conditions were used: 20 mL/min flow rate; 15 mingradients and a 20 mm x 50 mm Prism RP column with 5 micron particles(Thermo Hypersil-Keystone, Bellefonte, Pa.). For larger scalepurifications (typically greater than 100 mg crude sample), thefollowing conditions were used: 60 mL/min flow rate; 30 min gradientsand a 41.4 mm×250 mm Microsorb BDS column with 10 micron particles(Varian, Palo Alto, Calif.).

Example A Preparation of 2,2-Diphenyl-2-(S)-pyrrolidin-3-ylacetamideStep A: Preparation of (S)-1-Benzyl-3-(p-toluenesulfonyloxy)pyrrolidine

To a stirred solution of (S)-1-benzyl-3-pyrrolidinol (44.3 g, 0.25 mol)and 1,4-diazabicyclo[2.2.2]octane (33.7 g, 0.3 mol) in 250 mL oftert-butyl methyl ether under an atmosphere of nitrogen at 0° C., wasadded p-toluenesulfonyl chloride (52.4 g, 0.275 mol) portion-wise over20 minutes. The reaction mixture was stirred at 0° C. for 1 hour. Theice bath was removed and the mixture was stirred at ambient temperatureovernight (20±5 h). EtOAc (100 mL) was added, followed by saturatedaqueous sodium bicarbonate solution (250 mL). The resulting mixture wasstirred at ambient temperature for 1 hour. The layers were separated andthe organic layer was washed with saturated aqueous sodium bicarbonatesolution (250 mL); saturated aqueous ammonium chloride solution (250mL); saturated aqueous NaCl solution (250 mL); and then dried oversodium sulfate (80 g). The sodium sulfate was filtered off and washedwith EtOAc (20 mL) and the solvent was removed in vacuo to give 78.2 gof the title intermediate as an off-white solid (94% yield).

HPLC analysis of this intermediate was conducted using a YMC ODSA C184.6×50 mm column, having a 5.0 micron particle size. Detection was by UVabsorbance at 220 nm. The mobile phases employed were as follows (byvolume): A is MeOH (10%), water (90%) and TFA (0.1%); and B is MeOH(90%), water (10%) and TFA (0.1%). Using a flow rate of 4.0 mL/min of 0to 100% B in A over 5 minutes, this intermediate was determined to havea purity of 95%.

Step B: Preparation of(S)-1-Benzyl-3-(1-cyano-1,1-diphenylmethyl)-pyrrolidine

To a stirred solution of diphenylacetonitrile (12.18 g, 61.8 mmol) inanhydrous THF (120 mL) at 0° C., potassium tert-butoxide (10.60 g, 94.6mmol) was added over 5 minutes. The reaction mixture was stirred at 0°C. for 1 hour. To the reaction mixture at 0° C. was added(S)-1-benzyl-3-(p-toluenesulfonyloxy)-pyrrolidine (20.48 g, 61.3 mmol)in one portion. The cold bath was removed and the reaction mixture wasstirred for 5-10 min at which time the reaction mixture had become abrown homogeneous solution. The reaction mixture was then heated at 40°C. overnight (20±5 h). The reaction mixture (bright yellow suspension)was allowed to cool to ambient temperature before adding water (150 mL).Most of the THF was then removed in vacuo and IPAc (200 mL) was added.The layers were separated and the organic layer was washed withsaturated aqueous ammonium chloride solution (150 mL); saturated aqueousNaCl solution (150 mL); and then dried over sodium sulfate (50 g). Thesodium sulfate was filtered off and washed with IPAc (20 mL) and thesolvent was removed in vacuo to give 23.88 g of the title intermediateas a light brown oil (>99% yield). This intermediate was determined tohave a purity of 75% (contaminated mainly with excessdiphenylacetonitrile) using the HPLC method described in Step A.

Step C: Preparation of (S)-3-(1-Cyano-1,1-diphenylmethyl)pyrrolidine

(S)-1-Benzyl-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine was dissolved inIPAc (approximately 1 g/10 mL) and the solution was mixed with an equalvolume of 1N aqueous HCl. The resulting layers were separated and theaqueous layer was extracted with an equal volume of IPAc. The organiclayers were combined, dried over sodium sulfate and filtered. Thesolvent was removed in vacuo to afford(S)-1-benzyl-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine hydrochloride asa light yellow foamy solid. (Note: This hydrochloride salt can also beprepared during the work-up of Step B).

To a stirred solution of(S)-1-benzyl-3-(1-cyano-1,1-diphenylmethyl)-pyrrolidine hydrochloride(8.55 g, 21.98 mmol) in MeOH (44 mL) was added palladium on carbon (1.71g) and ammonium formate (6.93 g, 109.9 mmol). The reaction mixture washeated to 50° C. with stirring for 3 hours. The reaction was cooled toambient temperature and water (20 mL) was added. The resulting mixturewas filtered through a pad of Celite, washing with MeOH (20 mL). Thefiltrate was collected and most of the MeOH was removed in vacuo. Theresidue was mixed with IPAc (100 mL) and 10% aqueous sodium carbonate(50 mL). The resulting layers were separated and the aqueous layer wasextracted with IPAc (50 mL). The organic layers were combined and driedover sodium sulfate (20 g). The sodium sulfate was filtered off andwashed with IPAc (20 mL). The solvent was removed in vacuo to afford5.75 g of the title intermediate as a light yellow oil (99.7% yield, 71%purity by HPLC).

Step D: Preparation of 2,2-Diphenyl-2-(S)-pyrrolidin-3-ylacetamide

A 200 mL flask with a magnetic stir bar and a nitrogen inlet was chargedwith (S)-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine (2.51 g) and 80%H₂SO₄ (19.2 mL; pre-prepared with 16 mL of 96% H₂SO₄ and 3.2 mL of H₂O).The reaction mixture was then heated at 90° C. for 24 hours or until thestarting material was consumed as indicated by HPLC. The reactionmixture was allowed to cool to ambient temperature and then poured ontoice (approximately 50 mL by volume). A 50% aqueous NaOH solution wasadded slowly to the mixture with stirring over an ice bath until the pHwas about 12. DCM (200 mL) was added and mixed with the aqueous solutionat which time sodium sulfate precipitated out and was filtered off. Thefiltrate was collected and the layers were separated. The aqueous layerwas extracted with DCM (100 mL) and the organic layers were combined anddried with over sodium sulfate (5 g). The sodium sulfate was filteredoff and washed with DCM (10 mL). The solvent was removed in vacuo togive the crude product as a light yellow foamy solid (approximately 2.2g, 86% purity by HPLC).

The crude product was dissolved in EtOH (18 mL) with stirring. To thissolution was added a warm solution of L-tartaric acid (1.8 g) in EtOH(14 mL) and the resulting mixture was stirred overnight (15±5 h). Theresulting precipitate was isolated by filtration to give an off-whitesolid (approximately 3.2 g, >95% purity by HPLC). MeOH (15 mL) was addedto this solid and the resulting slurry was stirred at 70° C. overnight(15 h). The slurry was allowed to cool to ambient temperature and awhite solid (˜2.6 g, >99% purity by HPLC) was obtained after filtration.To this solid was added EtOAc (30 mL) and 1 N aqueous NaOH (25 mL). Thismixture was mixed until two distinct layers formed and then the layerswere separated and the aqueous layer was extracted with EtOAc (20 mL).The organic layers were combined and dried over sodium sulfate (10 g).The sodium sulfate was removed by filtration and the solvent wasevaporated in vacuo to afford 1.55 g of the title intermediate as anoff-white foamy solid (58% yield).

HPLC analysis was conducted using an Inertsil OCD-2 C18 column.Detection was by UV absorbance at 254 nm. The mobile phases employedwere as follows (by volume): A is MeOH (5%), water (95%), and TFA(0.1%); and B is MeOH (95%), water (5%) and TFA (0.1%). Using a flowrate of 1.0 mL/min of 0 to 100% B in A over 15 minutes, thisintermediate was determined to have a purity of >99%.

Example 1 Synthesis of2-{(S)-1-[5-(2-Methylamino)acetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Step A: Preparation of [(5-Hydroxypentylcarbamoyl)methyl]methylcarbamicAcid tert-Butyl Ester

5-Amino-1-pentanol (9.91 g, 96.0 mmol, 1.1 eq) in DCM (125 mL) was addeddropwise to N-tert-butoxycarbonylsarcosine hydroxysuccinimide ester (25g, 87.3 mmol, 1 eq) dissolved in DCM (50 mL) in a 500 mL flask and thereaction mixture was stirred at room temperature for 18 hours. Thesolution was then transferred to a separatory funnel and the organiclayer was washed with 1M sodium hydrogen sulfate (3×200 mL), 20%potassium carbonate (2×200 mL), brine (1×200 mL), dried over magnesiumsulfate, filtered and concentrated to yield 23.27 g of the titleintermediate as a semi-solid. ¹H NMR (CDCl₃): δ 6.3 (doublet, broad,1H), 3.85 (singlet, sharp, 2H), 3.65 (doublet of doublets, broad, 2h),3.3 (doublet of doublets, broad, 2h), 2.95 (singlet, sharp, 3H), 2.45(broad, 1H), 1.55 (multiplet, 4H), 1.45 (multiplet, 11H).

Step B: Preparation of Toluene-4-sulfonic Acid5-[2-(N′-tert-Butoxycarbonyl-N′-methylamino)acetylamino]pentyl Ester

To a stirred solution of[(5-hydroxypentylcarbamoyl)methyl]methylcarbamic acid tert-butyl ester(23.27 g, 84.8 mmol) in MTBE (100 mL) was added DABCO (12.37 g, 110.2mmol, 1.3 eq). To this mixture was added dropwise a solution of tosylchloride (19.4 g, 102 mmol, 1.2 eq) in MTBE (70 mL). The reactionmixture was stirred at room temperature for 18 hours and then filteredand the solids washed with MTBE. The organic phase was concentrated togive a clear oil, which was purified by silica gel flash chromatographyeluting with 2-5% MeOH in chloroform. The appropriate fractions werecombined to provide 22.7 g of the title intermediate as a colorless oil.¹H NMR (CDCl₃): δ 7.75 (doublet, sharp, 2H), 7.35 (doublet, sharp, 2H),6.1 (doublet, broad, 1H), 4.00 (triplet, sharp, 2H), 3.80 (singlet,sharp, 2H), 3.25 (doublet of doublets, broad, 2h), 2.95 (singlet, sharp,3H), 2.45 (singlet, sharp, 3H), 1.7 (multiplet, 2H), 1.40 (multiplet,13H).

Step C: Preparation of({5-[(S)-3-(Carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentylcarbamoyl}methyl)methylcarbamicAcid tert-Butyl Ester

To a stirred solution of toluene-4-sulfonic acid5-[2-(N′-tert-butoxycarbonyl-N′-methyl-amino)acetylamino]pentyl ester(1.37 g, 4.67 mmol, 1 eq) in ACN (10 mL) was added DIPEA (900 μL, 5.14mmol, 1.1 eq), followed by 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide,the intermediate from Example A (1.37 g, 4.90 mmol, 1.05 eq). Thereaction was stirred at 50° C. for 18 hours and then cooled to roomtemperature and concentrated to dryness. The obtained residue wasdissolved in a 2:1 solution of EtOAc and DCM (50 mL). The organic phasewas washed with 1M sodium phosphate (4×30 ML), 20% potassium carbonate(2×30 mL), brine (1×30 mL), dried over magnesium sulfate, filtered andconcentrated to give a light yellow foam. The crude material waspurified by silica gel flash chromatography eluting with 7.5% MeOH inchloroform containing 0.75% ammonium hydroxide to 10% MeOH in chloroformcontaining 1% ammonium hydroxide. The appropriate fractions werecombined to provide 1.42 g of the title intermediate as a white foam. MSm/z [M+H⁺] calc'd for C₃₁H₄₄N₄O₂ 537.3; found 537.3.

Step D: Preparation of2-{(S)1-[5-(2-Methylaminoacetylamino)pentyl]-pyrrolidin3-yl}-2,2-diphenylacetamide

To a stirred solution of({5-[(S)-3-(carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentylcarbamoyl}methyl)methylcarbamicacid tert-butyl ester (23.47 g, 43.7 mmol) in DCM (60 mL) was added 4 NHCl in dioxane (100 mL). The reaction mixture was stirred at roomtemperature for 2 hours and then concentrated to dryness. The residuewas dissolved in water (100 mL) and this solution was washed with DCM(4×60 mL). The aqueous phase was then made basic with 20% aqueouspotassium carbonate and extracted with DCM (4×60 mL). The combinedorganic phase was dried over magnesium sulfate, filtered andconcentrated. The residue was then dissolved in water (50 mL) containingTFA (2.5 eq) and purified by HPLC to obtain 17.5 g of the title compoundas a bis(trifluoroacetate) salt. MS m/z [M+H+] calc'd for C₂₆H₃₆N₄O₂437.3; found 437.5.

Example 2 Synthesis of2-{(S)-1-[5-(2-Aminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Step A: Preparation of 1-Bromo-5-(Di-tert-butoxycarbonyl)aminopentane

A solution of di-tert-butyliminodicarboxylate (Aldrich) (3.20 g, 0.15mol) in DMF (140 mL) was cooled to 5° C. and sodium hydride (60% inmineral oil, 653 mg, 0.16 mol) was slowly added. The reaction mixturewas slowly warmed to room temperature and stirred for 3 hours. Asolution of 1,5-dibromopentane (2.0 mL, 0.15 mmol) in DMF was added tothe reaction mixture. The reaction mixture was stirred at roomtemperature for 12 hours and then concentrated under vacuum and dilutedwith EtOAc (50 mL). The organic phase was washed with saturated sodiumbicarbonate (3×50 mL), brine (50 mL), dried over magnesium sulfate,filtered and concentrated under vacuum to provide 5.2 g of the titleintermediate as a white solid. MS m/z [M+Na⁺] calc'd for C₁₅H₂₈BrNO₄.Na388.1; found 388.0

Step B: Preparation of2-[(S)-1-(5-[Di-tert-butoxycarbonyl]aminopentyl)-pyrrolidin-3-yl]-2,2-diphenylacetamide

To a stirred solution of the intermediate from Step A above (1.29 g, 3.5mmol) and DIPEA (0.620 mL, 3.5 mmol) in DMF (10 mL) was added theintermediate of Example A, 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide(495 mg, 1.7 mmol). The resulting mixture was stirred for 48 hours andthen concentrated under vacuum. The residue was diluted with EtOAc andthe organic layer was washed with saturate sodium bicarbonate (2×10 mL),brine (1×10 mL), dried over magnesium sulfate, filtered and concentratedunder vacuum to give 1.2 g of the title intermediate. MS m/z [M+H⁺]calc'd for C₃₃H₄₇N₃O₅ 566.3; found 566.4.

Step C: Preparation of2-[(S)-1-(5-Aminopentyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

TFA (5 mL) was added to a solution of the intermediate from Step B (1.2g, 2.1 mmol) in DCM (5 mL). The mixture was stirred at room temperaturefor 2 hours and then concentrated under vacuum. The residue was dilutedwith DCM (10 mL) and the organic layer was washed with 1N NaOH (10 mL),saturated sodium bicarbonate (10 mL), dried over magnesium sulfate,filtered and concentrated under vacuum to provide 760 mg of the titleintermediate. MS m/z [M+H⁺] calc'd for C₂₃H₃₁N₃O 366.3; found 366.5.

Step D: Preparation of2-{(S)-1-[5-(2-Aminoacetylamino)pentyl]-pyrrolidin-3-yl}-2,2-diphenylacetamide

A solution of the intermediate from Step C (0.035 g, 0.1 mmol),N-BOC-glycine (0.022 g, 0.1 mmol), and DIPEA (0.018 mL, 0.1 mmol) in DMF(0.500 mL) was stirred at room temperature for 15 minutes. HATU (0.038mg, 0.0001 mmol) was added and the resulting mixture was stirred for 12hours at room temperature and then concentrated under vacuum. To theresidue was added DCM (0.500 mL) and TFA (0.250 mL) and this mixture wasstirred for 2 hours and then concentrated. To the residue was added a1:1 solution of water (0.500 mL) and ACN (0.500 mL) and this mixture waspurified by HPLC to give 14.6 mg of the title compound as abis(trifluoroacetate) salt. MS m/z [M+H⁺] calc'd for C₂₅H₃₄N₄O₂ 423.3;found 423.5.

Example 3 Synthesis of 2-Amino-N-{5-[(S)-3-(carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentyl}propionamide

Following the procedure of Example 2 and substituting2-tert-butoxy-carbonylaminopropionic acid in place of N-BOC-glycine,52.1 mg of the title compound was prepared as a bis(trifluoroacetate)salt. MS m/z [M+H⁺] calc'd for C₂₆H₃₆N₄O₂ 437.3; found 437.6.

Example 4 Synthesis of Pyrrolidine-2-carboxylic Acid{5-[(S)-3-(Carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentyl}amide

Following the procedure of Example 2 and substitutingpyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester in place ofN-BOC-glycine, 50.6 mg of the title compound was prepared as abis(trifluoroacetate) salt. MS m/z [M+H⁺] calc'd for C₂₈H₃₈N₄O₂ 463.3;found 463.6.

Example 5 Synthesis ofN-{5-[(S)-3-(Carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentyl}-2-methylaminopropionamide

Following the procedure of Example 2 and substituting2-(tert-butoxycarbonylmethylamino)propionic acid (0.024 g, 0.1 mmol) inplace of N-BOC-glycine, 39.0 mg of the title compound was prepared as abis(trifluoroacetate) salt. MS m/z [M+H⁺] calc'd for C₂₇H₃₈N₄O₂ 451.3;found 451.6

Example 6 Synthesis of2-{(S)-1-[5-(2-Ethylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Step A: Preparation of2-{(S)-1-[5-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

A 200-mL three-necked flask was charged with2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide, the intermediate fromExample A (2.8 g, 10 mmol), N-(5-bromo-pentyl)phthalimide (3.55 g, 12mmol), triethylamine (4.27 mL, 30 mmol) and ACN (100 mL). The reactionmixture was stirred at 55° C. for 8 hours and then concentrated underreduced pressure. IPAc was added to the residue and this mixture waswashed with 0.5 N NaOH (2×100 mL), brine (1×100 mL), dried overmagnesium sulfate, filtered and concentrated to give 5 g of the titleintermediate (100% yield). MS m/z [M+H⁺] calc'd for C₃₁H₃₃N₃O₃ 496.3;found 496.4.

Step B: Preparation of2-[(S)-1-(5-Amino)pentylpyrrolidin-3-yl]-2,2-diphenylacetamide

To a 50-mL three-necked flask, equipped with a magnetic stir bar and anitrogen gas inlet, were added the intermediate from Step A (4.89 g, 9.8mmol) and MeOH (10 mL). A solution of hydrazine (1.54 mL, 49 mmol) inMeOH (5 mL) was slowly added and the reaction mixture was stirred atroom temperature for 2 hours. The reaction mixture was then filtered andthe precipitate was washed with DCM (3×100 mL). The organic phase wasthen washed with 0.5N NaOH (2×100 mL), brine (1×100 mL), dried overmagnesium sulfate, filtered and concentrated to provide 3.58 g of thetitle intermediate (100% yield). MS m/z [M+H⁺] calc'd for C₂₃H₃₁N₃O366.2; found 366.3.

Step C: Preparation of2-{(S)-1-[5-(2-Chloroacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

To a 200-mL three-necked flask equipped with a magnetic stir bar, anitrogen gas inlet and an addition funnel, were added 2-chloroacetylchloride (132 μL, 1.65 mmol) and DCM (70 mL). The reaction mixture wascooled to a temperature of from about 0° C. to about 10° C. and asolution of the intermediate from Step B (550 mg, 1.5 mmol) and DIPEA(314 μL, 1.2 eq, 1.8 mmol) in DCM (30 mL) were added slowly. Thereaction mixture was then stirred at room temperature for 2 hours. Thereaction mixture was washed with saturated NaHCO₃ (2×50 mL), brine (1×50mL), dried over magnesium sulfate, filtered and concentrated to provide680 mg of the title intermediate (100% yield). MS m/z [M+H⁺] calc'd forC₂₅H₃₂ClN₃O₂ 442.2; found 442.5.

Step D: Preparation of2-{(S)-1-[5-(2-Ethylaminoacetylamino)pentyl]-pyrrolidin-3-yl}-2,2-diphenylacetamide

To a stirred solution of the intermediate from Step C (44 mg, 0.1 mmol)in DMF (1 mL) were added ethylamine (100 μL) and sodium iodide (30 mg,0.15 mmol). The mixture was heated at 80° C. for 16 hours. The solventwas then removed under reduced pressure. To the residue was added a 1:1solution of acetic acid and water (1.0 mL) and this mixture was purifiedby HPLC to afford 11 mg of the title compound as a bis(trifluoroacetate)salt. MS m/z [M+H⁺] calc'd for C₂₇H₃₈N₄O₂ 451.3; found 451.2.

Example 7 Synthesis of2,2-Diphenyl-2-{(S)-1-[5-(2-propylaminoacetylamino)pentyl]pyrrolidin-3-yl}acetamide

Following the procedure of Example 6 above and substituting propylamine(100 μL) in place of ethylamine, 13.1 mg of the title compound wereprepared as a bis(trifluoroacetate) salt. MS m/z [M+H⁺] calc'd forC₂₈H₄₀N₄O₂ 465.3; found 465.2.

Example 8 Synthesis of2-((S)-1-{5-[2-(2-Methoxyethylamino)acetylamino]pentyl}pyrrolidin-3-yl)-2,2-diphenylacetamide

Following the procedure of Example 6 and substituting2-methoxyethylamine (11.25 mg, 0.15 mmol) and DIPEA (35 μL, 0.2 mmol) inplace of ethylamine, 13.0 mg of the title compound were prepared as abis(trifluoroacetate) salt. MS m/z [M+H⁺] calc'd for C₂₈H₄₀N₄O₃ 481.3;found 481.2.

Example 9 Synthesis of2-{(S)-1-[5-(2-Benzylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Following the procedure of Example 6 and substituting benzylamine (16.1mg, 0.15 mmol) and DIPEA (35 μL, 0.2 mmol) in place of ethylamine, 14.5mg of the title compound were prepared as a bis(trifluoroacetate) salt.MS m/z [M+H⁺] calc'd for C₃₂H₄₀N₄O₂ 513.3; found 513.2.

Example 10 Synthesis of2-{(S)-1-[5-(2-Dimethylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Following the procedure of Example 2 and substitutingN,N-dimethylaminoacetic acid in place of N-BOC-glycine, the titlecompound was synthesized as a bis(trifluoroacetate) salt. MS m/z [M+H⁺]calc'd for C₂₇H₃₈N₄O₂ 451.3; found 451.3.

Example 11 Synthesis of2-{(S)-1-[5-(2-Cyclopropylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Following the procedure of Example 6 and substituting cyclopropylamine(8.6 mg, 0.15 mmol) and DIPEA (35 μL, 0.2 mmol) in place of ethylamine,16.9 mg of the title compound were prepared as a bis(trifluoroacetate)salt. MS m/z [M+H⁺] calc'd for C₂₈H₃₈N₄O₂ 463.3; found 463.2.

Example 12 Synthesis of2-{(S)-1-[5-(2-Cyclobutylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Following the procedure of Example 6 and substituting cyclobutylamine(10.7 mg, 0.15 mmol) and DIPEA (35 μL, 0.2 mmol) in place of ethylamine,13.7 mg of the title compound were synthesized as abis(trifluoroacetate) salt. MS m/z [M+H⁺] calc'd for C₂₉H₄₀N₄O₂ 477.3;found 477.2.

Example 13 Synthesis of2-{(S)-1-[5-(2-Cyclopentylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide

Following the procedure of Example 6 and substituting cyclopentylamine(12.8 mg, 0.15 mmol) and DIPEA (35 μL, 0.2 mmol) in place of ethylamine,14.6 mg of the title compound were prepared as a bis(trifluoroacetate)salt. MS m/z [M+H⁺] calc'd for C₃₀H₄₂N₄O₂ 491.3; found 491.2.

Assay 1 Radioligand Binding Assay A. Membrane Preparation from CellsExpressing hM₁, hM₂, hM₃ and hM₄ Muscarinic Receptor Subtypes

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in medium consisting of HAM's F-12 supplemented with 10% FBS(Fetal Bovine Serum) and 250 μg/mL Geneticin. The cells were grown in a5% CO₂, 37° C. incubator and lifted with 2 mM EDTA in dPBS. Cells werecollected by 5 minute centrifugation at 650×g, and cell pellets wereeither stored frozen at −80° C. or membranes were prepared immediately.For membrane preparation, cell pellets were resuspended in lysis bufferand homogenized with a Polytron PT-2100 tissue disrupter (Kinematica AG;20 seconds×2 bursts). Crude membranes were centrifuged at 40,000×g for15 minutes at 4° C. The membrane pellet was then resuspended withresuspension buffer and homogenized again with the Polytron tissuedisrupter. The protein concentration of the membrane suspension wasdetermined by the method described in Lowry, O. et al., Journal ofBiochemistry 193:265 (1951). All membranes were stored frozen inaliquots at −80° C. or used immediately. Aliquots of prepared hM₅receptor membranes were purchased directly from Perkin Elmer and storedat −80° C. until use.

B. Radioligand Binding Assay on Muscarinic Receptor Subtypes hM₁, hM₂,hM₃, hM₄ and hM₅

Radioligand binding assays were performed in 96-well microtiter platesin a total assay volume of 100 μL. CHO cell membranes stably expressingeither the hM₁, hM₂, hM₃, hM₄ or hM₅ muscarinic subtype were diluted inassay buffer to the following specific target protein concentrations(μg/well): 10 μg for hM₁, 10-15 μg for hM2, 10-20 μg for hM₃, 10-20 μgfor hM₄, and 10-12 μg for hM₅. The membranes were briefly homogenizedusing a Polytron tissue disruptor (10 seconds) prior to assay plateaddition. Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 400 μM in dilution buffer and then serially diluted5× with dilution buffer to final concentrations ranging from 10 μM to100 μM. The addition order and volumes to the assay plates were asfollows: 25 μL radioligand, 25 μL diluted test compound, and 50 μLmembranes. Assay plates were incubated for 60 minutes at 37° C. Bindingreactions were terminated by rapid filtration over GF/B glass fiberfilter plates (PerkinElmer Inc., Wellesley, Mass.) pre-treated in 1%BSA. Filter plates were rinsed three times with wash buffer (10 mMHEPES) to remove unbound radioactivity. Plates were then air dried, and50 μL Microscint-20 liquid scintillation fluid (PerkinElmer Inc.,Wellesley, Mass.) was added to each well. The plates were then countedin a PerkinElmer Topcount liquid scintillation counter (PerkinElmerInc., Wellesley, Mass.). Binding data were analyzed by nonlinearregression analysis with the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.) using the one-site competition model.K_(i) values for test compounds were calculated from observed IC₅₀values and the K_(D) value of the radioligand using the Cheng-Prusoffequation (Cheng Y; Prusoff W. H. Biochemical Pharmacology22(23):3099-108 (1973)). K_(i) values were converted to pK_(i) values todetermine the geometric mean and 95% confidence intervals. These summarystatistics were then converted back to K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. Exemplary compoundsof the invention that were tested in this assay, were found to have aK_(i) value of less than about 100 nM for the M₃ muscarinic receptorsubtype in this assay. For example, the compounds of Examples 1 to 9were found to have K_(i) values of less than 50 nM for the M₃ muscarinicreceptor.

Assay 2 Muscarinic Receptor Functional Potency Assays A. Blockade ofAgonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound was determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor. cAMP assays were performed ina radioimmunoassay format using the Flashplate Adenylyl CyclaseActivation Assay System with ¹²⁵I-cAMP (NEN SMP004B, PerkinElmer LifeSciences Inc., Boston, Mass.), according to the manufacturer'sinstructions.

Cells were rinsed once with dPBS and lifted with Trypsin-EDTA solution(0.05% trypsin/0.53 mM EDTA) as described in the Cell Culture andMembrane Preparation section above. The detached cells were washed twiceby centrifugation at 650×g for five minutes in 50 mLs DPBS. The cellpellet was then re-suspended in 10 mL dPBS, and the cells were countedwith a Coulter Z1 Dual Particle Counter (Beckman Coulter, Fullerton,Calif.). The cells were centrifuged again at 650×g for five minutes andre-suspended in stimulation buffer to an assay concentration of1.6×10⁶-2.8×10⁶ cells/mL.

The test compound was initially dissolved to a concentration of 400 μMin dilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and thenserially diluted with dilution buffer to final molar concentrationsranging from 100 μM to 0.1 nM. Oxotremorine was diluted in a similarmanner.

To measure oxotremorine inhibition of adenylyl cyclase (AC) activity, 25μL forskolin (25 μM final concentration diluted in dPBS), 25 μL dilutedoxotremorine, and 50 μL cells were added to agonist assay wells. Tomeasure the ability of a test compound to block oxotremorine-inhibitedAC activity, 25 μL forskolin and oxotremorine (25 μM and 5 μM finalconcentrations, respectively, diluted in dPBS) 25 μL diluted testcompound, and 50 μL cells were added to remaining assay wells.

Reactions were incubated for 10 minutes at 37° C. and stopped byaddition of 100 μL ice-cold detection buffer. Plates were sealed,incubated overnight at room temperature and counted the next morning ona PerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) wascalculated based on the counts observed for the samples and cAMPstandards, as described in the manufacturer's user manual. Data wereanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(i), using the EC₅₀ of theoxotremorine concentration-response curve and the oxotremorine assayconcentration as the K_(D) and [L], respectively. The K_(i) values wereconverted to pK_(i) values to determine the geometric mean and 95%confidence intervals. These summary statistics were then converted backto K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Exemplary compoundsof the invention that were tested in this assay, typically were found tohave a K_(i) value of less than about 100 nM for blockade ofoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor. For example, the compound ofExample 1 was found to have a K_(i) value of less than about 100 nM.

B. Blockade of Agonist-Mediated [³⁵S]GTPγS Binding

In a second functional assay, the fimctional potency of test compoundscan be determined by measuring the ability of the compounds to blockoxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor.

At the time of use, frozen membranes were thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes were briefly homogenized using a PolytronPT-2100 tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine wasdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following was added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine(EC₉₀) and GDP (3 μM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates were then incubated at 37° C. for 60 minutes. The assayplates were filtered over 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates were rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) was added to each well, and each plate wassealed and radioactivity counted on a topcounter (PerkinElmer). Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(i), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Exemplary compoundsof the invention that were tested in this assay, typically were found tohave a K_(i) value of less than about 100 nM for blockade ofoxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor. For example, in this assay the compound of Example 1was found to have a K_(i) value of less than about 100 nM.

C. Blockade of Agonist-Mediated Calcium Release via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event was measuredin real time by the FLIPR, which detected the change in fluorescencefrom a monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency was determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM₁, hM₃ and cM₅ receptors were seeded into 96-well FLIPR plates thenight before the assay was done. Seeded cells were washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in Hank's Buffered SaltSolution (HBSS) without calcium and magnesium) to remove growth mediaand leaving 50 μL/well of FLIPR buffer. The cells were then incubatedwith 50 μL/well of 4 μM FLUO-4AM (a 2× solution was made) for 40 minutesat 37° C., 5% carbon dioxide. Following the dye incubation period, cellswere washed two times with FLIPR buffer, leaving a final volume of 50μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine was first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells were first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which is performed by the FLIPR. An EC₉₀ value for oxotremorine wasgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100−F){circumflex over ( )}1/H)*EC₅₀. An oxotremorineconcentration of 3×EC_(F) is prepared in stimulation plates such that anEC₉₀ concentration of oxotremorine was added to each well in theantagonist inhibition assay plates.

The parameters used for the FLIPR were: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline was determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measured thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence was expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data was analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K_(i) values weredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Exemplary compoundsof the invention that were tested in this assay, in this assay typicallywere found to have a K_(i) value of less than about 100 μM for blockadeof agonist-mediated calcium release in CHO cells stably expressing thehM₃ receptor. For example, in this assay the compound of Example 1 wasfound to have a K_(i) value of less than about 100 nM for the hM₃receptor.

Assay 3 Determination of Duration of Bronchoprotection in Guinea PigModel of Acetylcholine-Induced Bronchoconstriction

This in vivo assay was used to assess the bronchoprotective effects oftest compounds exhibiting muscarinic receptor antagonist activity.

Groups of six male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g were individuallyidentified by cage cards. Throughout the study animals were allowedaccess to food and water ad libitum.

Test compounds were administered via inhalation over 10 minutes in awhole-body exposure dosing chamber (R&S Molds, San Carlos, Calif.). Thedosing chambers were arranged so that an aerosol was simultaneouslydelivered to 6 individual chambers from a central manifold. Guinea pigswere exposed to an aerosol of a test compound or vehicle (WFI). Theseaerosols were generated from aqueous solutions using an LC StarNebulizer Set (Model 22F51, PARI Respiratory Equipment, Inc. Midlothian,Va.) driven by a mixture of gases (CO₂=5%, O₂=21% and N₂=74%) at apressure of 22 psi. The gas flow through the nebulizer at this operatingpressure was approximately 3 L/minute. The generated aerosols weredriven into the chambers by positive pressure. No dilution air was usedduring the delivery of aerosolized solutions. During the 10 minutenebulization, approximately 1.8 mL of solution was nebulized. This wasmeasured gravimetrically by comparing pre-and post-nebulization weightsof the filled nebulizer.

The bronchoprotective effects of test compounds administered viainhalation were evaluated using whole body plethysmography at 1.5, 24,48 and 72 hours post-dose.

Forty-five minutes prior to the start of the pulmonary evaluation, eachguinea pig was anesthetized with an intramuscular injection of ketamine(43.75 mg/kg), xylazine (3.50 mg/kg) and acepromazine (1.05 mg/kg).After the surgical site was shaved and cleaned with 70% alcohol, a 2-3cm midline incision of the ventral aspect of the neck was made. Then,the jugular vein was isolated and cannulated with a saline-filledpolyethylene catheter (PE-50, Becton Dickinson, Sparks, Md.) to allowfor intravenous infusions of ACh (Sigma-Aldrich, St. Louis, Mo.) insaline. The trachea was then dissected free and cannulated with a 14 Gteflon tube (#NE-014, Small Parts, Miami Lakes, Fla.). If required,anesthesia was maintained by additional intramuscular injections of theaforementioned anesthetic mixture. The depth of anesthesia was monitoredand adjusted if the animal responded to pinching of its paw or if therespiration rate was greater than 100 breaths/minute.

Once the cannulations were complete, the animal was placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula (PE-160, Becton Dickinson, Sparks, Md.) wasinserted to measure pulmonary driving pressure (pressure). The teflontracheal tube was attached to the opening of the plethysmograph to allowthe guinea pig to breathe room air from outside the chamber. The chamberwas then sealed. A heating lamp was used to maintain body temperatureand the guinea pig's lungs were inflated 3 times with 4 mL of air usinga 10 mL calibration syringe (#5520 Series, Hans Rudolph, Kansas City,Mo.) to ensure that the lower airways did not collapse and that theanimal did not suffer from hyperventilation.

Once it was determined that baseline values were within the range0.3-0.9 mL/cm H₂O for compliance and within the range 0.1-0.199 cmH₂O/mL per second for resistance, the pulmonary evaluation wasinitiated. A Buxco pulmonary measurement computer progam enabled thecollection and derivation of pulmonary values.

Starting this program initiated the experimental protocol and datacollection. The changes in volume over time that occur within theplethysmograph with each breath were measured via a Buxco pressuretransducer. By integrating this signal over time, a measurement of flowwas calculated for each breath. This signal, together with the pulmonarydriving pressure changes, which were collected using a Sensym pressuretransducer (#TRD4100), was connected via a Buxco (MAX 2270) preamplifierto a data collection interface (#'s SFT3400 and SFT3813). All otherpulmonary parameters were derived from these two inputs.

Baseline values were collected for 5 minutes, after which time theguinea pigs were challenged with ACh. ACh (0.1 mg/mL) was infusedintravenously for 1 minute from a syringe pump (sp210iw, World PrecisionInstruments, Inc., Sarasota, Fla.) at the following doses and prescribedtimes from the start of the experiment: 1.9 μg/minute at 5 minutes, 3.8μg/minute at 10 minutes, 7.5 μg/minute at 15 minutes, 15.0 μg/minute at20 minutes, 30 μg/minute at 25 minutes and 60 μg/minute at 30 minutes.If resistance or compliance had not returned to baseline values at 3minutes following each ACh dose, the guinea pig's lungs were inflated 3times with 4 mL of air from a 10 mL calibration syringe. Recordedpulmonary parameters included respiration frequency (breaths/minute),compliance (mL/cm H₂O) and pulmonary resistance (cm H₂O/mL per second).Once the pulmonary function measurements were completed at minute 35 ofthis protocol, the guinea pig was removed from the plethysmograph andeuthanized by carbon dioxide asphyxiation.

The data were evaluated in one or both of the following ways:

-   -   (a) Pulmonary resistance (R_(L), cm H₂O/mL per second) was        calculated from the ratio of “change in pressure” to “the change        in flow.” The _(RL) response to ACh (60 μg/min, IH) was computed        for the vehicle and the test compound groups. The mean ACh        response in vehicle-treated animals, at each pre-treatment time,        was calculated and used to compute % inhibition of ACh response,        at the corresponding pre-treatment time, at each test compound        dose. Inhibition dose-response curves for ‘R_(L)’ were fitted        with a four parameter logistic equation using GraphPad Prism,        version 3.00 for Windows (GraphPad Software, San Diego, Calif.)        to estimate bronchoprotective ID₅₀ (dose required to inhibit the        ACh (60 μg/min) bronchoconstrictor response by 50%). The        equation used was as follows:        Y=Min+(Max−Min)/(1+10^(((log ID50−X)*Hillslope)))        where X is the logarithm of dose, Y is the response (%        Inhibition of ACh induced increase in R_(L)). Y starts at Min        and approaches asymptotically to Max with a sigmoidal shape.    -   (b) The quantity PD₂, which is defined as the amount of ACh or        histamine needed to cause a doubling of the baseline pulmonary        resistance, was calculated using the pulmonary resistance values        derived from theflow and the pressure over a range of ACh or        histamine challenges using the following equation (which is        derived from a equation used to calculate PC₂₀ values described        in American Thoracic Society. Guidelines for methacholine and        exercise challenge testing—1999. Am J Respir Crit Care Med. 161:        309-329 (2000)):        ${PD}_{2} = {{antilog}\lbrack {{\log\quad C_{1}} + \frac{( {{\log\quad C_{2}} - {\log\quad C_{1}}} )( {{2R_{0}} - R_{1}} )}{R_{2} - R_{1}}} \rbrack}$        where:    -   C₁=concentration of ACh or histamine preceding C₂    -   C₂=concentration of ACh or histamine resulting in at least a        2-fold increase in pulmonary resistance (R_(L))    -   R₀=Baseline R_(L) value    -   R₁=R_(L) value after C₁    -   R₂=R_(L) value after C₂

An efficacious dose was defined as a dose that limited thebronchrestriction response to a 50 μg/mL dose of ACh to a doubling ofthe baseline pulmonary resistance(PD_(2(50)). Statistical analysis of the data was performed using a two-tailed Students t-test. A P-value<)0.05was considered significant.

Generally, test compounds having a PD_(2(50) less than about) 200 μg/mLfor ACh-induced bronchoconstriction at 1.5 hours post-dose in this assayare preferred. For example, the compound of Example 1 was found to havea PD_(2(50) less than about) 200 μg/mL for ACh-inducedbronchoconstriction at 1.5 hours post-dose.

Assay 4 Inhalation Guinea Pig Salivation Assay

Guinea pigs (Charles River, Wilmington, Mass.) weighing 200-350 g wereacclimated to the in-house guinea pig colony for at least 3 daysfollowing arrival. Test compound or vehicle were dosed via inhalation(IH) over a 10 minute time period in a pie shaped dosing chamber (R&SMolds, San Carlos, Calif.). Test solutions were dissolved in sterilewater and delivered using a nebulizer filled with 5.0 mL of dosingsolution. Guinea pigs were restrained in the inhalation chamber for 30minutes. During this time, guinea pigs were restricted to an area ofapproximately 110 sq. cm. This space was adequate for the animals toturn freely, reposition themselves, and allow for grooming. Following 20minutes of acclimation, guinea pigs were exposed to an aerosol generatedfrom a LS Star Nebulizer Set (Model 22F51, PARI Respiratory Equipment,Inc. Midlothian, Va.) driven by house air at a pressure of 22 psi. Uponcompletion of nebulization, guinea pigs were evaluated at 1.5, 6, 12,24, 48, or 72 hrs after treatment.

Guinea pigs were anesthetized one hour before testing with anintramuscular (IM) injection of a mixture of ketamine 43.75 mg/kg,xylazine 3.5 mg/kg, and acepromazine 1.05 mg/kg at an 0.88 mL/kg volume.Animals were placed ventral side up on a heated (37° C.) blanket at a 20degree incline with their head in a downward slope. A 4-ply 2×2 inchgauze pad (Nu-Gauze General-use sponges, Johnson and Johnson, Arlington,Tex.) was inserted in the guinea pig's mouth. Five minutes later, themuscarinic agonist pilocarpine (3.0 mg/kg, SC) was administered and thegauze pad was immediately discarded and replaced by a new pre-weighedgauze pad. Saliva was collected for 10 minutes, at which point the gauzepad was weighed and the difference in weight recorded to determine theamount of accumulated saliva (in mg). The mean amount of salivacollected for animals receiving the vehicle and each dose of testcompound was calculated. The vehicle group mean was considered to be100% salivation. Results were calculated using result means (n=3 orgreater). Confidence intervals (95%) were calculated for each dose ateach time point using two-way ANOVA. This model is a modified version ofthe procedure described in Rechter, “Estimation of anticholinergic drugeffects in mice by antagonism against pilocarpine-induced salivation”Ata Pharmacol Toxicol 24:243-254 (1996).

The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, was calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose. Theinhibition dose-response data were fitted to a four parameter logisticequation using GraphPad Prism, version 3.00 for Windows (GraphPadSoftware, San Diego, Calif.) to estimate anti-sialagogue ID₅₀ (doserequired to inhibit 50% of pilocarpine-evoked salivation). The equationused was as follows:Y=Min+(Max−Min)/(1+10^(((log ID50−X)*Hilislope)))where X is the logarithm of dose, Y is the response (% inhibition ofsalivation). Y starts at Min and approaches asymptotically to Max with asigmoidal shape.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ was usedto compute the apparent lung selectivity index of the test compound.Generally, compounds having an apparent lung selectivity index greaterthan about 5 are preferred. In this assay, the compound of Example 1 hadan apparent lung-selectivity index greater than about 5.

Assay 5 Methacholine-Induced Depressor Responses in Conscious GuineaPigs

Healthy, adult, male Sprague-Dawley guinea pigs (Harlan, Indianapolis,Ind.), weighing between 200 and 300 g were used in these studies. Underisoflurane anesthesia (to effect), animals were instrumented with commoncarotid artery and jugular vein catheters (PE-50 tubing). The catheterswere exteriorized utilizing a subcutaneous tunnel to the subscapulararea. All surgical incisions were sutured with 4-0 Ethicon Silk and thecatheters locked with heparin (1000 units/mL). Each animal wasadministered saline (3 mL, SC) at the end of surgery as well asbuprenorphine (0.05 mg/kg, IM). Animals were allowed to recover on aheating pad before being returned to their holding rooms.

Approximately 18 to 20 hours following surgery, the animals were weighedand the carotid artery catheter on each animal was connected to atransducer for recording arterial pressure. Arterial pressure and heartrate was recorded using a Biopac MP-100 Acquisition System. Animals wereallowed to acclimate and stabilize for a period of 20 minutes.

Each animal was challenged with MCh (0.3 mg/kg, IV) administered throughthe jugular venous line and the cardiovascular response was monitoredfor 10 minutes. The animals were then placed into the whole body dosingchamber, which was connected to a nebulizer containing the test compoundor vehicle solution. The solution was nebulized for 10 minutes using agas mixture of breathable air and 5% carbon dioxide with a flow rate of3 liters/minute. The animals were then removed from the whole bodychamber and returned to their respective cages. At 1.5 and 24 hpost-dosing, the animals were re-challenged with MCh (0.3 mg/kg, IV) andthe hemodynamic response was determined. Thereafter, the animals wereeuthanized with sodium pentobarbital (150 mg/kg, IV).

MCh produces a decrease in mean arterial pressure (MAP) and decrease inheart rate (bradycardia). The peak decrease, from baseline, in MAP(depressor responses) was measured for each MCh challenge (before andafter IH dosing). The bradycardic effects were not used for analysissince these responses were not robust and reproducible. The effects oftreatment on the MCh responses are expressed as % inhibition (mean ±SEM)of the control depressor responses. Two-way ANOVA with the appropriatepost-hoc test was used to test the effects of treatment andpre-treatment time. The depressor responses to MCh were relativelyunchanged at 1.5 and 24 h after inhalation dosing with vehicle.

The ratio of the anti-depressor ID₅₀ to bronchoprotective ID₅₀ was usedto compute apparent lung-selectivity of the test compound. Generally,compounds having an apparent lung-selectivity index greater than 5 arepreferred. In this assay, the compound of Example 1 had an apparentlung-selectivity index greater than 5.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

1. A compound of formula I:

wherein: each R¹ and R² are independently selected from (1-4C)alkyl,(2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(a),—SR^(a), —NR^(a)R^(b), —S(O)R^(c) and —S(O)₂R^(c); where each R^(a) andR^(b) independently represents hydrogen, (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl or (3-6C)cycloalkyl; each R^(c) independently represents(1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl or (3-6C)cycloalkyl; or twoadjacent R¹ groups or two adjacent R² groups are joined together to form(3-6C)alkylene, (2-4C)alkylene-O— or —O-(2-4C)alkylene-O—; a and b eachindependently are 0 or an integer of from 1 to 5; each R³ independentlyis fluoro or (1-4C)alkyl; c is 0 or an integer of from 1 to 3; R^(4a)and R^(4b) are independently selected from hydrogen, (1-4C)alkyl, andphenyl-(1-4C)alkyl; or R^(4a) and R^(4b) together with the carbon atomto which they are attached form a (3-6C)heterocyclic ring optionallycontaining one additional heteroatom selected from nitrogen, oxygen orsulfur and wherein the heterocyclic ring is unsubstituted or substitutedwith 1 or 2 substituents selected independently from (1-4C)alkyl andfluoro; e is 1 or2; m is 4, 5 or 6; R⁵ is selected from hydrogen,(1-4C)alkyl, and (3-4C)cycloalkyl; R⁶ is hydrogen or an unbranched(1-4C)alkyl; or R⁶ and R⁸ are joined, together with the atoms to whichthey are attached, to form a pyrrolidin-2-yl group; R⁷ is selected fromhydrogen, (1-6C)alkyl, (3-6C)cycloalkyl, —CH₂Ar¹, —CH₂CH₂—OH and—CH₂CH₂—O-(1-4C)alkyl; wherein Ar¹ represents phenyl or(3-5C)heteroaryl, wherein the phenyl or heteroaryl group isunsubstituted or substituted with from 1 to 3 substituents selectedindependently from halo, (1-4C)alkyl and (1-4C)alkoxy; wherein eachalkyl and alkoxy is optionally substituted with from 1 to 3 fluorosubstituents; and R⁸ is hydrogen or (1-6C)alkyl; or R⁷ and R₈ togetherwith the nitrogen atom to which they are attached form apyrrolidin-1-yl, piperidin-1-yl, morpholin-1-yl or thiomorpholin-1-ylgroup; wherein each alkyl group in R¹, R², R³, R^(4a), R^(4b), R⁵, R⁶,R⁷, R⁸ and R^(a-c) is optionally substituted with from 1 to 5 fluorosubstituents; or a pharmaceutically acceptable salt or solvate orstereoisomer thereof.
 2. The compound of claim 1, wherein a, b and ceach represents
 0. 3. The compound of claim 1, wherein R^(4a) and R^(4b)are hydrogen.
 4. The compound of claim 1, wherein e is
 1. 5. Thecompound of claim 1, wherein m is
 5. 6. The compound of claim 1, whereinR⁵ is hydrogen.
 7. The compound of claim 1, wherein R⁶ is hydrogen or anunbranched (1-4C)alkyl.
 8. The compound of claim 1, wherein a, b, and care 0; R^(4a) and R^(4b) are hydrogen; e is 1; m is 5; and R⁵ ishydrogen.
 9. The compound of claim 1, wherein a, b, and c are 0; R^(4a)and R^(4b) are hydrogen; e is 1; m is 5; R⁵ is hydrogen; and R⁶ ishydrogen
 10. The compound of any one of claims 1 to 9, wherein R⁷ isselected from hydrogen, (1-4C)alkyl, (3-5C)cycloalkyl, —CH₂Ar¹, and—CH₂CH₂—O-(1-3C)alkyl, where Ar¹ represents phenyl.
 11. The compound ofany one of claims 1 to 9, wherein R⁸ is hydrogen or (1-4C)alkyl.
 12. Acompound selected from:2-{(S)-1-[5-(2-methylamino)acetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[5-(2-aminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-amino-N-{5-[(S)-3-(carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentyl}propionamide;pyrrolidine-2-carboxylic acid{5-[(S)-3-(carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentyl}amide;N-{5-[(S)-3-(carbamoyldiphenylmethyl)pyrrolidin-1-yl]pentyl}-2-methylaminopropionamide;2-{(S)-1-[5-(2-ethylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2,2-diphenyl-2-{(S)-1-[5-(2-propylaminoacetylamino)pentyl]pyrrolidin-3-yl}acetamide;2-((S)-1-{5-[2-(2-methoxyethylamino)acetylamino]pentyl}pyrrolidin-3-yl)-2,2-diphenylacetamide;2-{(S)-1-[5-(2-benzylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[5-(2-dimethylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[5-(2-cyclopropylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;2-{(S)-1-[5-(2-cyclobutylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;and2-{(S)-1-[5-(2-cyclopentylaminoacetylamino)pentyl]pyrrolidin-3-yl}-2,2-diphenylacetamide;or a pharmaceutically acceptable salt or solvate thereof.
 13. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of claim 1or
 12. 14. The pharmaceutical composition of claim 13, wherein thecomposition further comprises a therapeutically effective amount of anagent selected from β₂ adrenergic receptor agonists, steroidalanti-inflammatory agents, phosphodiesterase-4 inhibitors, andcombinations thereof.
 15. The pharmaceutical composition of claim 14,wherein the composition comprises a therapeutically effective amount ofa β₂ adrenergic receptor agonist and a steroidal anti-inflammatoryagent.
 16. A process for preparing a compound of claim 1 or 12, theprocess comprising: (a) reacting a compound of formula II:

with a compound of formula III:X¹—(CH₂)_(m)—NR⁵CO—CHR⁶—NR⁷R⁸   III wherein X¹ represents a leavinggroup; (b) coupling a compound of formula IV:

with a compound of formula V:HOOC—CHR⁶—NR⁷R⁸   V or a reactive derivative thereof; (c) reacting acompound of formula II with a compound of formula VI:OHC—(CH₂)_(m-1)—NR⁵CO—CHR⁶—NR⁷R⁸   VI in the presence of a reducingagent; or (d) for a compound of formula (I) in which R⁶ represents ahydrogen atom or an unbranched (1-4C)alkyl group, reacting a compound offormula VII:

wherein X² represents a leaving group, with a compound of formula VIII:HNR⁷R⁸   VIII to provide a compound of formula I.
 17. The process ofclaim 16, wherein the process further comprises forming apharmaceutically acceptable salt of the compound of formula I.
 18. Theproduct prepared by the process of claim
 16. 19. The product prepared bythe process of claim
 17. 20. A method of studying a biological system orsample comprising a muscarinic receptor, the method comprising: (a)contacting the biological system or sample with a compound of claim 1 or12; and (b) determining the effects caused by the compound on thebiological system or sample.
 21. A method for antagonizing a muscarinicreceptor in a mammal which comprises administering to the mammal, atherapeutically effective amount of a compound of claim 1 or
 12. 22. Amethod for treating a pulmonary disorder, the method comprisingadministering to a patient a therapeutically effective amount of acompound of claim 1 or
 12. 23. A method of producing bronchodilation ina patient, the method comprising administering to a patient abronchodilation-producing amount of a compound of claim 1 or
 12. 24. Amethod of treating chronic obstructive pulmonary disease or asthma, themethod comprising administering to a patient a therapeutically effectiveamount of a compound of claim 1 or 12.